Plants with improved agronomic traits

ABSTRACT

Isolated polynucleotides and polypeptides and recombinant DNA constructs useful for conferring drought tolerance, compositions (such as plants or seeds) comprising these recombinant DNA constructs, and methods utilizing these recombinant DNA constructs. The recombinant DNA construct comprises a polynucleotide operably linked to a promoter that is functional in a plant, wherein said polynucleotide encodes a PRE2 polypeptide.

CROSS-REFERENCE

This continuation application is a continuation application of U.S. Ser.No. 13/819,619 filed Feb. 27, 2013, which is a 35 USC §371 NationalStage application of PCT/US2012/44434 filed Jun. 27, 2012, which claimspriority to U.S. Ser. No. 61/503,852 filed Jul. 1, 2011, all of whichare incorporated herein by reference.

FIELD

The field of disclosure relates to plant breeding and genetics and, inparticular, relates to recombinant DNA constructs useful in plants forconferring improved agronomic traits.

BACKGROUND

Improving agronomic traits in crop plants is beneficial to farmers.Several factors crop yield. Abiotic stress is the primary cause of croploss worldwide, causing average yield losses of more than 50% for majorcrops. Among the various abiotic stresses, drought is a major factorthat limits crop productivity worldwide. Exposure of plants to awater-limiting environment during various developmental stages appearsto activate various physiological and developmental changes. Molecularmechanisms of abiotic stress responses and the genetic regulatorynetworks of drought stress tolerance have been studied.

Natural responses to abiotic stress vary among plant species and amongvarieties and cultivars within a plant species. Certain species,varieties or cultivars are more tolerant to abiotic stress such asdrought than others. Transgenic approaches including overexpression anddownregulation are evaluated for engineering drought tolerance in cropplants. Nitrogen utilization efficiency also affects crop yield,especially where the application of nitrogen fertilizer is limited.

SUMMARY

Methods and compositions to increase yield and stress tolerance inplants are disclosed. In an embodiment, reduced activity or expressionof Pre2 gene results in increased tolerance to drought and improvednitrogen utilization.

A method of altering an agronomic trait or parameter of a plant, themethod includes expressing a polynucleotide that down-regulates theendogenous expression of a messenger RNA encoding a polypeptide, whereinthe polypeptide includes a conserved domain selected from the groupconsisting of SEQ ID NOS: 27-48. In an embodiment, the agronomic traitor parameter is selected from the group consisting of drought tolerance,increased nitrogen use efficiency, and increased yield. In anembodiment, the suppression of endogenous expression of the messengerRNA is by RNAi.

In an embodiment, the expression of the endogenous Pre2 gene orproduction of its protein is reduced by anti-sense expression,co-suppression, dsRNA, ribozymes, microRNA, genome editing, targetedpromoter inactivation, site-directed mutagenesis and knock-outs.

In an embodiment, a plant comprising in its genome a recombinant DNAconstruct comprising a polynucleotide operably linked to at least oneregulatory element, wherein said polynucleotide comprises a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence encoding a polypeptide with drought tolerance activity, whereinthe polypeptide has an amino acid sequence of at least 60%, 80%, 85%,90%, 95% or 100% sequence identity, based on the Clustal W method ofalignment with pairwise alignment default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=0.2, DELAY DEVERGENT SEQS(%)=30%, DNA TRANSITIONWEIGHT=0.5, PROTEIN WEIGHT MATRIX “Gonnet Series”), when compared to asequence selected from the group consisting of SEQ ID NOS: 3, 5, 7, 9,11, 13, 15, 18, 20 and 22; (b) a nucleotide sequence encoding apolypeptide with drought tolerance activity, wherein the nucleotidesequence is hybridizable under stringent conditions with a DNA moleculecomprising the full complement to a sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21,23-26; (c) a nucleotide sequence encoding a polypeptide with droughttolerance activity, wherein the nucleotide sequence is derived from asequence selected from the group consisting of SEQ ID NOS: 1, 2, 4, 6,8, 10, 12, 14, 16, 17, 19, 21, 23-26 by alteration of one or morenucleotides by at least one method selected from the group consistingof: deletion, substitution, addition and insertion; (d) a nucleotidesequence encoding a polypeptide wherein the amino acid sequence of thepolypeptide comprises a sequence selected from the group consisting ofSEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; and (e) a nucleotidesequence comprising a sequence selected from the group consisting of SEQID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; and whereinsaid plant exhibits increased drought tolerance when compared to acontrol plant not comprising said recombinant DNA construct. The plantmay be a monocot or dicot.

In another embodiment, a plant comprising in its genome a recombinantDNA construct comprising a polynucleotide operably linked to at leastone regulatory element, wherein said polynucleotide comprises anucleotide sequence selected from the group consisting of: (a) anucleotide sequence encoding a polypeptide with drought toleranceactivity, wherein the polypeptide has an amino acid sequence of at least60%, 80%, 85%, 90%, 95% or 100% sequence identity, based on the ClustalW method of alignment with pairwise alignment default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=0.2, DELAY DEVERGENT SEQS(%)=30%, DNATRANSITION WEIGHT=0.5, PROTEIN WEIGHT MATRIX “Gonnet Series”), whencompared to a sequence selected from the group consisting of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; (b) a nucleotide sequenceencoding a polypeptide with drought tolerance activity, wherein thenucleotide sequence is hybridizable under stringent conditions with aDNA molecule comprising the full complement to a sequence selected fromthe group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17,19, 21, 23-26; (c) a nucleotide sequence encoding a polypeptide withdrought tolerance activity, wherein the nucleotide sequence is derivedfrom a sequence selected from the group consisting of SEQ ID NOS: 1, 2,4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26 by alteration of one or morenucleotides by at least one method selected from the group consistingof: deletion, substitution, addition and insertion; (d) a nucleotidesequence encoding a polypeptide wherein the amino acid sequence of thepolypeptide comprises a sequence selected from the group consisting ofSEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; and (e) a nucleotidesequence comprising a sequence selected from the group consisting of SEQID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; and whereinsaid plant exhibits an increase in yield when compared to a controlplant not comprising said recombinant DNA construct. The plant mayexhibit said increase in yield when compared, under water limitingconditions, to said control plant not comprising said recombinant DNAconstruct. The plant may be a monocot or dicot.

In another embodiment, a method of increasing drought tolerance in aplant, comprising: (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory element, wherein said polynucleotide comprises anucleotide sequence selected from the group consisting of: (i) anucleotide sequence encoding a polypeptide with drought toleranceactivity, wherein the polypeptide has an amino acid sequence of at least60%, 80%, 85%, 90%, 95% or 100% sequence identity, based on the ClustalW method of alignment with pairwise alignment default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=0.2, DELAY DEVERGENT SEQS(%)=30%, DNATRANSITION WEIGHT=0.5, PROTEIN WEIGHT MATRIX “Gonnet Series”), whencompared to a sequence selected from the group consisting of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; (ii) a nucleotide sequenceencoding a polypeptide with drought tolerance activity, wherein thenucleotide sequence is hybridizable under stringent conditions with aDNA molecule comprising the full complement to a sequence selected fromthe group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17,19, 21, 23-26; (iii) a nucleotide sequence encoding a polypeptide withdrought tolerance activity, wherein the nucleotide sequence is asequence selected from the group consisting of SEQ ID NOS: 1, 2, 4, 6,8, 10, 12, 14, 16, 17, 19, 21, 23-26 by alteration of one or morenucleotides by at least one method selected from the group consistingof: deletion, substitution, addition and insertion; (iv) a nucleotidesequence encoding a polypeptide wherein the amino acid sequence of thepolypeptide comprises a sequence selected from the group consisting ofSEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; and (v) a nucleotidesequence comprising a sequence selected from the group consisting of SEQID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; and (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct and exhibits increased drought tolerance whencompared to a control plant not comprising the recombinant DNAconstruct. The method may further comprise: (c) obtaining a progenyplant derived from the transgenic plant, wherein said progeny plantcomprises in its genome the recombinant DNA construct and exhibitsincreased drought tolerance when compared to a control plant notcomprising the recombinant DNA construct.

In another embodiment, a method of evaluating drought tolerance in aplant, comprising: (a) obtaining a transgenic plant, wherein thetransgenic plant comprises in its genome a recombinant DNA constructcomprising a polynucleotide operably linked to at least one regulatoryelement, wherein said polynucleotide comprises a nucleotide sequenceselected from the group consisting of: (i) a nucleotide sequenceencoding a polypeptide with drought tolerance activity, wherein thepolypeptide has an amino acid sequence of at least 60%, 80%, 85%, 90%,95% or 100% sequence identity, based on the Clustal W method ofalignment with pairwise alignment default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=0.2, DELAY DEVERGENT SEQS(%)=30%, DNA TRANSITIONWEIGHT=0.5, PROTEIN WEIGHT MATRIX “Gonnet Series”), when compared to asequence selected from the group consisting of SEQ ID NOS: 3, 5, 7, 9,11, 13, 15, 18, 20 and 22; (ii) a nucleotide sequence encoding apolypeptide with drought tolerance activity, wherein the nucleotidesequence is hybridizable under stringent conditions with a DNA moleculecomprising the full complement of a sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21,23-26; (iii) a nucleotide sequence encoding a polypeptide with droughttolerance activity, wherein the nucleotide sequence is a sequenceselected from the group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12,14, 16, 17, 19, 21, 23-26 by alteration of one or more nucleotides by atleast one method selected from the group consisting of: deletion,substitution, addition and insertion; (iv) a nucleotide sequenceencoding a polypeptide wherein the amino acid sequence of thepolypeptide comprises a sequence selected from the group consisting ofSEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; and (v) a nucleotidesequence comprising a sequence selected from the group consisting of SEQID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; and (b)obtaining a progeny plant derived from the transgenic plant of (a),wherein the progeny plant comprises in its genome the recombinant DNAconstruct; and (c) evaluating the progeny plant for drought tolerancecompared to a control plant not comprising the recombinant DNAconstruct.

In another embodiment, a method of determining an alteration of anagronomic characteristic in a plant, comprising: (a) obtaining atransgenic plant, wherein the transgenic plant comprises in its genome arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory element, wherein said polynucleotide comprises anucleotide sequence selected from the group consisting of: (i) anucleotide sequence encoding a polypeptide with drought toleranceactivity, wherein the polypeptide has an amino acid sequence of at least60%, 80%, 85%, 90%, 95% or 100% sequence identity, based on the ClustalW method of alignment with pairwise alignment default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=0.2, DELAY DEVERGENT SEQS(%)=30%, DNATRANSITION WEIGHT=0.5, PROTEIN WEIGHT MATRIX “Gonnet Series”), whencompared to a sequence selected from the group consisting of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; (ii) a nucleotide sequenceencoding a polypeptide with drought tolerance activity, wherein thenucleotide sequence is hybridizable under stringent conditions with aDNA molecule comprising the full complement of a sequence selected fromthe group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17,19, 21, 23-26; (iii) a nucleotide sequence encoding a polypeptide withdrought tolerance activity, wherein the nucleotide sequence is asequence selected from the group consisting of SEQ ID NOS: 1, 2, 4, 6,8, 10, 12, 14, 16, 17, 19, 21, 23-26 by alteration of one or morenucleotides by at least one method selected from the group consistingof: deletion, substitution, addition and insertion; (iv) a nucleotidesequence encoding a polypeptide wherein the amino acid sequence of thepolypeptide comprises a sequence selected from the group consisting ofSEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; and (v) a nucleotidesequence comprising a sequence selected from the group consisting of SEQID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; and (b)obtaining a progeny plant derived from the transgenic plant of step (a),wherein the progeny plant comprises in its genome the recombinant DNAconstruct; and (c) determining whether the progeny plant exhibits analteration of at least one agronomic characteristic when compared to acontrol plant not comprising the recombinant DNA construct. Saiddetermining step (c) may comprise determining whether the transgenicplant exhibits an alteration of at least one agronomic characteristicwhen compared, under water limiting conditions, to a control plant notcomprising the recombinant DNA construct. Said at least one agronomictrait may be yield and furthermore may be an increase in yield.

In another embodiment, an isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence encoding a polypeptide with drought toleranceactivity, wherein the polypeptide has an amino acid sequence of at least60%, 80%, 85%, 90% or 95% sequence identity, based on the Clustal Wmethod of alignment with pairwise alignment default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=0.2, DELAY DEVERGENT SEQS(%)=30%, DNATRANSITION WEIGHT=0.5, PROTEIN WEIGHT MATRIX “Gonnet Series”), whencompared to a sequence selected from the group consisting of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; b) a nucleotide sequence encodinga polypeptide with drought tolerance activity, wherein the nucleotidesequence is hybridizable under stringent conditions with a DNA moleculecomprising the full complement of a sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21,23-26; (c) a nucleotide sequence encoding a polypeptide with droughttolerance activity, wherein the nucleotide sequence is a sequenceselected from the group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12,14, 16, 17, 19, 21, 23-26 by alteration of one or more nucleotides by atleast one method selected from the group consisting of: deletion,substitution, addition and insertion; (d) a nucleotide sequence encodinga polypeptide wherein the amino acid sequence of the polypeptidecomprises a sequence selected from the group consisting of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; and (e) a a nucleotide sequencecomprising a sequence selected from the group consisting of SEQ ID NOS:1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26.

In another embodiment, an isolated polynucleotide comprising the fullcomplement of the nucleotide sequence of the disclosure, wherein thefull complement and the nucleotide sequence of the disclosure consist ofthe same number of nucleotides and are 100% complementary.

In another embodiment, a recombinant DNA construct comprising theisolated polynucleotide of the disclosure operably linked to at leastone regulatory element.

In another embodiment, a cell comprising the recombinant DNA constructof the disclosure, wherein the cell is selected from the groupconsisting of a bacterial cell, a yeast cell, and insect cell and aplant cell.

In another embodiment, a plant or a seed comprising the recombinant DNAconstruct of the disclosure. The plant or seed may be a monocot or adicot plant or seed.

In another embodiment, a method for isolating a polypeptide encoded bythe recombinant DNA construct of the disclosure, wherein the methodcomprises the following: (a) transforming a cell with the recombinantDNA construct of the disclosure; (b) growing the transformed cell ofstep (a) under conditions suitable for expression of the recombinant DNAconstruct; and (c) isolating the polypeptide from the transformed cellof step (b).

In another embodiment, an isolated polypeptide selected from the groupconsisting of: (a) a polypeptide with drought tolerance activity,wherein the polypeptide has an amino acid sequence of at least 60%, 80%,85%, 90% or 95% sequence identity, based on the Clustal W method ofalignment with pairwise alignment default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=0.2, DELAY DEVERGENT SEQS(%)=30%, DNA TRANSITIONWEIGHT=0.5, PROTEIN WEIGHT MATRIX “Gonnet Series”), when compared to asequence selected from the group consisting of SEQ ID NOS: 3, 5, 7, 9,11, 13, 15, 18, 20 and 22; (b) a polypeptide with drought toleranceactivity, wherein the amino acid sequence is a sequence selected fromthe group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17,19, 21, 23-26; by alteration of one or more amino acids by at least onemethod selected from the group consisting of: deletion, substitution,addition and insertion; and (c) a polypeptide wherein the amino acidsequence of the polypeptide comprises a sequence selected from the groupconsisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20 and 22.

In another embodiment, a vector that includes the polynucleotide of thedisclosure is described.

In another embodiment, a method for producing a transgenic plantcomprising transforming a plant cell with the recombinant DNA constructof the disclosure and regenerating a transgenic plant from thetransformed plant cell.

In another embodiment, the present disclosure includes any of the plantsof the present disclosure wherein the plant is selected from the groupconsisting of: maize, soybean, sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, millet, sugarcane, switchgrass, tobacco,potato and sugar beet.

In another embodiment, the present disclosure includes any of themethods of the present disclosure wherein the plant is selected from thegroup consisting of: maize, soybean, sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, millet, sugarcane, switchgrass, tobacco,potato and sugar beet.

In another embodiment, the present disclosure includes seed of any ofthe plants of the present disclosure, wherein said seed comprises in itsgenome a recombinant DNA construct comprising a polynucleotide operablylinked to at least one regulatory element, wherein said polynucleotideencodes a polypeptide having an amino acid sequence of at least 60%sequence identity, based on the Clustal W method of alignment, whencompared to a sequence selected from the group consisting of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; and wherein a plant produced fromsaid seed exhibits either an increased drought tolerance, or an increasein yield, or both, when compared to a control plant not comprising saidrecombinant DNA construct.

A method of identifying a plant that exhibits increased droughttolerance or an improved agronomic parameter, the method includesscreening a population of maize plants for drought tolerance or enhancednitrogen utilization efficiency and analyzing the sequence of apolynucleotide encoding a protein comprising SEQ ID NO: 3 andidentifying the plant with drought tolerance or enhanced nitrogenutilization efficiency.

A method of identifying alleles in maize plants or germplasm that areassociated with enhanced tolerance to drought and/or increased nitrogenuse efficiency comprising:

-   -   (a) obtaining a population of maize plants, wherein one or more        plants exhibit differing levels of enhanced tolerance to drought        and/or increased nitrogen use efficiency;    -   (b) evaluating allelic variations with respect to the        polynucleotide sequence encoding a protein comprising SEQ ID NO:        3 or in the genomic region that regulates the expression of the        polynucleotide encoding the protein;    -   (c) obtaining phenotypic values of enhanced tolerance to drought        and/or increased nitrogen use efficiency for a plurality of        maize plants in the population;    -   (d) associating the allelic variations in the genomic associated        with SEQ ID NO: 1 with said tolerance; and    -   (e) identifying the alleles that are associated with enhanced        tolerance.

A transgenic plant includes in its genome a recombinant construct, therecombinant construct comprising a genetic element that reduces theexpression of an endogenous gene, wherein the endogenous gene encodes apolypeptide that comprises an amino acid sequence of SEQ ID NO: 3 or asequence that is 90% identical to SEQ ID NO: 3. In an embodiment, thegenetic element includes a RNAi construct.

A plant comprising in its genome a genetic modification that results inthe reduced expression of a gene that encodes a polypeptide thatcomprises an amino acid sequence of SEQ ID NO: 3 or a sequence that is95% identical to SEQ ID NO: 3 or the reduced activity of thepolypeptide, wherein the plant shows one or more improved agronomicparameters that contribute to drought tolerance or yield. In anembodiment, the plant is a maize plant.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

The disclosure can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1 shows the phenotype of pre-mature senescence (pre2) mutation (1A)and co-segregation analysis using SAIFF (Selective Amplification ofInsertion Flanking Fragments) protocol to isolate a candidate generesponsible for the pre2 mutant phenotype in corn (1B).

FIG. 2 shows RT-PCR and Southern blot analyses of Pre2 candidate gene:Reverse transcriptase-polymerase chain reaction (RT-PCR) of pre2 mutantshowing four transcripts with variable intensities as compared to one inWT-sib (2A). Cloning and sequence analysis of these transcripts were dueto interference of the Mutator resulting into differential splicing inmutant mature RNA (2C). Southern blot analysis of pre2-2 mutant alleleindicates a tight linkage between the pre2 mutant phenotype with thepolymorphism in the candidate gene (2B).

FIG. 3 shows a diagramatic representation of gene expression of Pre2gene in different plant parts of Arabodopsis. Red (dark shade) andyellow (light shade) colors depict the highest and lowest geneexpression, respectively.

FIG. 4 show PCR fingerprinting of T-DNA insertion plants of Arabidopsis:PCR-FP analysis to identify homozygous knockouts, heterozygous andwild-type plants for T-DNA insertion of pre2 gene.

FIG. 5A shows that homozygous (pre2/pre2) knockout mutants (#11 and #25)exhibit robust growth and more siliques at flowering as compared to itsboth wild-type (+/+) and heterozygous (+/pre2) sibs. FIG. 5B showsaverage biomass of homozygous T-DNA knockout mutant (KO) plants issignificantly higher as compared to its homozygous WT-sibs (WT) andheterozygous WT-sibs (Het).

FIG. 6 shows Arabidopsis knockout mutant (homozygous) for corn homologof pre2 candidate gene and its WT-sib were screened for drought assay.The Atpre2 mutant was an outlier in this assay with a score (2 sigma)higher than 0.9 and with positive deviation. Arabidopsis transgene withcorn native gene was a control and was hypersensitive to drought stress.

FIG. 7 shows phenotypic response of Arabidopsis knockout mutant(homozygous) for homolog of corn pre2 along with its WT-sib screened onLow N.

FIG. 8 shows screening of pre2 knockout mutant for pre2 of Arabidopsisshowing root inhibition (sensitivity) to high N.

FIG. 9 shows trait summary of T0 plants for ear characteristics and seednumber along with their molecular analysis (A) and the T1 reproductiveassays results for three events (B). Significantly positive attributesare shown in bold.

FIG. 10 (A-E) shows multiple alignment of Arabidopsis Pre2 peptide withmonocots (Bahia, Sudan and Resurrection grasses, sorghum, rice, andmaize) and other dicots (soybean and castor bean). The order ofsequences shown in the alignment is SEQ ID NOS: 15, 9, 5, 22, 18, 20, 3,13, 7 and 11. The consensus regions are shown at the end of thealignment. A few exemplary conserved regions are indicated by horizontalbars.

FIG. 11(A-C) shows conserved domain sequences from Pre2 polypeptidesequences.

FIG. 12 shows germination rate on media containing 1 μM ABA. Col-0 andAtpre2 are represented as dark and light boxes, respectively. The dataare averages of germination rate with standard deviations from threereplications.

SUMMARY OF SEQ ID NOS

Description and Abbreviation SEQ ID NO. Maize (ZmPre2 genomic sequence)1 Maize (ZmPre2 cDNA sequence) 2 Maize (ZmPRE2 amino acid sequence) 3Rice (OsPre2 cDNA) 4 Rice (OsPRE2 aa sequence) 5 Sorghum (SbPre2 cDNA) 6Sorghum (SbPRE2_aa sequence) 7 BahiaGrass cDNA sequence 8 BahiaGrassPRE2 aa 9 SudanGrass_CDS (partial length sequence) 10 SudanGrass_aapartial length sequence 11 ResurrectionGrass CDS 12 ResurrectionGrass_aa13 At1g72390FL cDNA Arabidopsis 14 AtPRE2_aa sequence 15At1g72390genomic Arabidopsis 16 GM_chr16_Pre2 CDS 17 GM_chr16_Pre2(amino acid) 18 GM_chr7_Pre2 (CDS) 19 GM_chr7_Pre2 (amino acid) 20Castor bean Pre2 CDS 21 Castor bean PRE2 amino acid 22BrassicaOleracea_Pre2 23 (gi_17734666_gb_BH526581.1) CDSBrassicaRapa_Pre2(PBR136351) CDS 24 Canola(PBN029307) CDS 25 SoybeanGM-Pre2 (PSO423639) DNA 26 ZmPre2 TR1 (Fwd) 49 ZmPre2 TR1 (Rev) 50Soybean Pre2 RNAi target sequence 51 Conserved Domain 1 27 ConservedDomain 2 28 Conserved Domain 3 29 Conserved Domain 4 30 Conserved Domain5 31

Description and SEQ Abbreviation Consensus sequences (amino acid) ID NO:Conserved Region 1 MSLENIVKDIPSISDNSWTYGDLMEVESKILKALQPK 32 LHLDPTPKLDRLConserved Region 2 SWTYGDLMEVES 33 Conserved Region 3SWTYGDLMEVESKILKALQP 34 Conserved Region 4 GKKVCIDRVQESS 35Conserved Region 5 QSPRLSAGALPQSPLSSKSGEFS 36 Conserved Region 6SPLSSKSGEFS 37 Conserved Region 7 AQLAAKRRSNSLPKT 38 Conserved Region 8VGSPVSVGTTSVPLNANSP 39 Conserved Region 9 RFSKIEMVTMRHQLNFKK 40Conserved Region 10 LPNTHSADLLAQQFCSLMVREG 41 Conserved Region 11QALQMSQGLLSGVSM 42 Conserved Region 12SPQQMSQRTPMSPQISSGAIHAMSAGNPEACPASP 43 QLSSQTLGSVSSITNSPMConserved Region 13 CPASPQLSSQTLGSVSSITNSPM 44 Conserved Region 14HEVSFTFSLYDRGYLISKSAAMDPSQTSIQDGKTLH 45PYDRASEKLFSAIEAGRLPGDILDEIPSKYYNGSVVCEIRDYRKHVSNQAPASSAELGLPIVNKVRLRMTFENVVKDITLLSDDSWSYRDFVEAEARIVRALQPELCLDPTPKLDRLCQDPVPHKLSLGIGKKRRLRQNPEVVVT SSNMSHGKKVCIDR Conserved Region 15LCLDPTPKLDRLCQDPVPHKLSLGIGKKRRLRQNP 46 Conserved Region 16 LCLDPTPKLDRL47 Conserved Region 17 QDPVPHKLSLGIGKKRRLRQNP 48

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825.

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J.219(2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION

Pre2 nucleotide sequences and polypeptide sequences improve stresstolerance and yield of agronomically important crop plants andvegetables. Reduced expression of Pre2 mRNA results in enhancedtolerance to drought and improved utilization of nitrogen (NUE) undernitrogen limiting conditions. Suppressing of one or more Pre2 endogenousgenes results in improved agronomic performance. One way of suppressingendogenous Pre2 gene expression is through RNAi. Other modes ofsuppression include anti-sense, co-suppression, promoter invertedrepeats, and micro RNA. Another non-transgenic approach is to generatenative variation in the expression levels of endogenous Pre2.

One or more of the plant Pre2 polypeptides disclosed herein include anSpt20 domain that is found in the Spt20 family of proteins from bothhuman and yeast. The Spt20 protein is part of the SAGA complex which isa large complex that may be involved in histone deacetylation. YeastSpt20 has been shown to play a role in structural integrity of the SAGAcomplex as no intact SAGA could be purified in spt20 deletion strains.The Spt20 domain or a sub-region thereof may be involved in DNA binding.For example, in an embodiment, the Spt20 domain comprises amino acidpositions 69-227 of the castor bean Pre2 polypeptide. Relative positionsin other Pre2 homologs or orthologs from one or more other species alsocontain this conserved region. In an embodiment, this conserved regionis designated as “pfam12090”.

Pre2 homozygous mutants were robust in growth with more pod numbers butwere late in maturity by 4 to 5 days as compared to its WT-sibs (FIG.5A). For measuring total biomass, 9 whole plants, each of knock out #11,knock out #25, homozygous WT, and heterozygous WT-sibs, were harvestedand air dried for 14 days at room temperature. Total weight wasdetermined by weighing and average and standard deviation werecalculated for statistical analysis. The total biomass of both knockouts(combined) was found to be significantly higher (t test at P<0.01) whencompared to both homozygous and heterozygous WT-sibs (FIG. 5B). In anembodiment, three maize gene suppression events (e.g., RNAi eventsnamely 1.4, 1.5, and 2.5) exhibited improved agronomic parameters in anNUE Reproductive Assay in T1 generation under 4.0 mMolNitrate-suboptimal nitrogen conditions. Two of three events (1.5 and2.5) showed significant increase (percent change vs. Null) in silkcount, ear length, ear width, and ear area (FIG. 9B). In addition tothese traits, event 2.5 also showed significant difference for Days toshed and days to silk as compared to its nulls. Thus, transgenic plantswhere the expression of the Pre2 mRNA has been modulated exhibitsignificant differences in one or more agronomic parameters of interestfor crop plants.

In ABA-sensitivity experiments, Atpre2 mutant showed a hypersensitiveresponse to ABA in a dosage dependent manner. The seed germination inmutant was reduced or delayed by more than 50% as compare to wild typein presence of 1 uM ABA (FIG. x). Endogenous AT-PRE2 gene expression washigher in guard cells in wild type plants and was down-regulated by ABAtreatment both in seedling and leaf based on gene expression databases.In addition AtPRE2 was also up-regulated by nitrate in roots. Theseresults indicate a direct or indirect role of AtPRE2 in ABA and Nsignaling/pathway.

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety. Some of the agronomic parameters thatcorrelate with nitrogen use efficiency analysis and/or include for e.g.,root dwt (g), root: shoot dwt ratio, shoot dwt (g), shoot nitrogen (mg/gdwt), shoot total nitrogen (mg) and total plant dwt (g). Some of thevariables that for nitrogen use efficiency reproductive assay includee.g., anthesis to silking interval (days), days to shed, days to silk,ear area 8 days after silk (sq cm), ear length 8 days after silk (cm),ear width 8 days after silk (cm), max total area, specific growth rate,and silk count.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein:

The terms “monocot” and “monocotyledonous plant” are usedinterchangeably herein. A monocot of the current disclosure includes theGramineae.

The terms “dicot” and “dicotyledonous plant” are used interchangeablyherein. A dicot of the current disclosure includes the followingfamilies: Brassicaceae, Leguminosae, and Solanaceae.

The terms “full complement” and “full-length complement” are usedinterchangeably herein, and refer to a complement of a given nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary.

“Arabidopsis” and “Arabidopsis thaliana” are used interchangeablyherein, unless otherwise indicated.

An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from acDNA library and therefore is a sequence which has been transcribed. AnEST is typically obtained by a single sequencing pass of a cDNA insert.The sequence of an entire cDNA insert is termed the “Full-InsertSequence” (“FIS”). A “Contig” sequence is a sequence assembled from twoor more sequences that can be selected from, but not limited to, thegroup consisting of an EST, FIS and PCR sequence. A sequence encoding anentire or functional protein is termed a “Complete Gene Sequence”(“CGS”) and can be derived from an FIS or a contig.

“Agronomic characteristic” or “agronomic parameter” is a measurableparameter including but not limited to, greenness, yield, growth rate,biomass, fresh weight at maturation, dry weight at maturation, fruityield, seed yield, total plant nitrogen content, fruit nitrogen content,seed nitrogen content, nitrogen content in a vegetative tissue, totalplant free amino acid content, fruit free amino acid content, seed freeamino acid content, free amino acid content in a vegetative tissue,total plant protein content, fruit protein content, seed proteincontent, protein content in a vegetative tissue, drought tolerance,nitrogen uptake, root lodging, harvest index, stalk lodging, plantheight, ear height, ear length, salt tolerance, early seedling vigor andseedling emergence under low temperature stress.

“Transgenic” refers to any cell, cell line, callus, tissue, plant partor plant, the genome of which has been altered by the presence of aheterologous nucleic acid, such as a recombinant DNA construct,including those initial transgenic events as well as those created bysexual crosses or asexual propagation from the initial transgenic event.The term “transgenic” as used herein does not encompass the alterationof the genome (chromosomal or extra-chromosomal) by conventional plantbreeding methods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition or spontaneousmutation.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. For example, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably and is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine and “N” for anynucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA that is without introns andthat can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from amRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase I.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or pro-peptides present in the primarytranslation product have been removed.

Nitrogen utilization efficiency (NUE) genes affect yield and haveutility for improving the use of nitrogen in crop plants, especiallymaize. Increased nitrogen use efficiency can result from enhanced uptakeand assimilation of nitrogen fertilizer and/or the subsequentremobilization and reutilization of accumulated nitrogen reserves, aswell as increased tolerance of plants to stress situations such as lownitrogen environments. The genes can be used to alter the geneticcomposition of the plants, rendering them more productive with currentfertilizer application standards or maintaining their productive rateswith significantly reduced fertilizer or reduced nitrogen availability.Improving NUE in corn would increase corn harvestable yield per unit ofinput nitrogen fertilizer, both in developing nations where access tonitrogen fertilizer is limited and in developed nations where the levelof nitrogen use remains high. Nitrogen utilization improvement alsoallows decreases in on-farm input costs, decreased use and dependence onthe non-renewable energy sources required for nitrogen fertilizerproduction and reduces the environmental impact of nitrogen fertilizermanufacturing and agricultural use

“Precursor” protein refers to the primary product of translation ofmRNA; i.e., with pre- and pro-peptides still present. Pre- andpro-peptides may be and are not limited to intracellular localizationsignals.

“Isolated” refers to materials, such as nucleic acid molecules and/orproteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acidfragments that are not normally found together in nature. Accordingly, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that normally found in nature.

The terms “entry clone” and “entry vector” are used interchangeablyherein.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include, but are not limited to,promoters, translation leader sequences, introns, and polyadenylationrecognition sequences. The terms “regulatory sequence” and “regulatoryelement” are used interchangeably herein.

“Promoter” refers to a nucleic acid fragment capable of controllingtranscription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably, and refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” refers to a promoter whose activityis determined by developmental events.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

“Expression” refers to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct) into a cell, means “transfection” or“transformation” or “transduction” and includes reference to theincorporation of a nucleic acid fragment into a eukaryotic orprokaryotic cell where the nucleic acid fragment may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation andtransient transformation.

“Stable transformation” refers to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentis stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

“Allele” is one of several alternative forms of a gene occupying a givenlocus on a chromosome. When the alleles present at a given locus on apair of homologous chromosomes in a diploid plant are the same thatplant is homozygous at that locus. If the alleles present at a givenlocus on a pair of homologous chromosomes in a diploid plant differ thatplant is heterozygous at that locus. If a transgene is present on one ofa pair of homologous chromosomes in a diploid plant that plant ishemizygous at that locus.

The percent identity between two amino acid or nucleic acid sequencesmay be determined by visual inspection and mathematical calculation.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the MEGALIGN® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the Clustal W method of alignment(Thompson, et al., (1994). Nucleic Acids Research 22:4673-80) with thedefault parameters (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, DELAYDEVERGENT SEQS(%)=30%, DNA TRANSITION WEIGHT=0.5, PROTEIN WEIGHT MATRIX“Gonnet Series”).

Default parameters for pairwise alignments using the Clustal W methodwere SLOW-ACCURATE, GAP PENALTY=10, GAP LENGTH=0.10, PROTEIN WEIGHTMATRIX “Gonnet 250”. After alignment of the sequences, using the ClustalW program, it is possible to obtain “percent identity” and “divergence”values by viewing the “sequence distances” table on the same program;unless stated otherwise, percent identities and divergences provided andclaimed herein were calculated in this manner.

Alternatively, sequence alignments and percent identity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the Clustal V methodof alignment (Higgins and Sharp, (1989) CAB/OS 5:151-153) with thedefault parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments and calculation of percent identityof protein sequences using the Clustal V method are KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids theseparameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.

Alternatively, the percent identity of two protein sequences may bedetermined by comparing sequence information based on the algorithm ofNeedleman and Wunsch, (J. Mol. Biol. 48:443-453, 1970) and using the GAPcomputer program available from the University of Wisconsin GeneticsComputer Group (UWGCG). The preferred default parameters for the GAPprogram include: (1) a scoring matrix, blosum62, as described byHenikoff and Henikoff, (Proc. Natl. Acad. Sci. USA 89:10915-10919 1992);(2) a gap weight of 12; (3) a gap length weight of 4; and (4) no penaltyfor end gaps.

Other programs used by those skilled in the art of sequence comparisonmay also be used. The percent identity can be determined by comparingsequence information using, e.g., the BLAST program described byAltschul, et al., (Nucl. Acids. Res. 25:3389-3402 1997). This program isavailable on the Internet at the web site of the National Center forBiotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ).The details of various conditions (parameters) for identity search usingthe BLAST program are shown on these web sites, and default values arecommonly used for search although part of the settings may be changed asappropriate. Alternatively, the percent identity of two amino acidsequences may be determined by using a program such as geneticinformation processing software GENETYX Ver.7 (Genetyx Corporation,Japan) or using an algorithm such as FASTA. In this case, default valuesmay be used for search.

The percent identity between two nucleic acid sequences can bedetermined by visual inspection and mathematical calculation, or morepreferably, the comparison is done by comparing sequence informationusing a computer program. An exemplary, preferred computer program isthe Genetic Computer Group (GCG®; Madison, Wis.) WISCONSIN PACKAGE®version 10.0 program, “GAP” (Devereux, et al., (1984) Nucl. Acids Res.12:387). In addition to making a comparison between two nucleic acidsequences, this “GAP” program can be used for comparison between twoamino acid sequences and between a nucleic acid sequence and an aminoacid sequence. The preferred default parameters for the “GAP” programinclude: (1) the GCG® implementation of a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted amino acid comparison matrix of Gribskovand Burgess, (1986) Nucl. Acids Res. 14:6745, as described by Schwartzand Dayhoff, eds., “Atlas of Polypeptide Sequence and Structure,”National Biomedical Research Foundation, pp. 353-358, (1979), or othercomparable comparison matrices; (2) a penalty of 30 for each gap and anadditional penalty of 1 for each symbol in each gap for amino acidsequences, or penalty of 50 for each gap and an additional penalty of 3for each symbol in each gap for nucleotide sequences; (3) no penalty forend gaps; and (4) no maximum penalty for long gaps. Other programs usedby those skilled in the art of sequence comparison can also be used,such as, for example, the BLASTN program version 2.2.7, available foruse via the National Library of Medicine website, or the WU-BLAST 2.0algorithm (Advanced Biocomputing, LLC). In addition, the BLAST algorithmuses the BLOSUM62 amino acid scoring matrix, and optional parametersthat can be used are as follows: (A) inclusion of a filter to masksegments of the query sequence that have low compositional complexity(as determined by the SEG program of Wootton and Federhen (Computers andChemistry, 1993); also see, Wootton and Federhen, (1996) MethodsEnzymol. 266:554-71) or segments consisting of short-periodicityinternal repeats (as determined by the XNU program of Claverie andStates (Computers and Chemistry, 1993)), and (B) a statisticalsignificance threshold for reporting matches against database sequences,or E-score (the expected probability of matches being found merely bychance, according to the stochastic model of Karlin and Altschul, 1990;if the statistical significance ascribed to a match is greater than thisE-score threshold, the match will not be reported); preferred E-scorethreshold values are 0.5, or in order of increasing preference, 0.25,0.1, 0.05, 0.01, 0.001, 0.0001, 1 e-5, le-10, 1 e-15, 1 e-20, 1 e-25, 1e-30, 1 e-40, 1 e-50, 1 e-75 or 1 e-100.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, etal., Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).

The term “consisting essentially of” in the context of a polypeptidesequence generally refers to the specified portion of the amino acidsequence and those other sequences that do not materially affect thebasic and novel characteristics of the disclosed sequences herein. Forexample, in the context of an RNAi sequence, the term consistingessentially generally refers to that portion of the target sequence andthose other nucleotide sequences that do not materially affect thebinding and suppressing properties of the sequence targets disclosedherein.

Embodiments include isolated polynucleotides and polypeptides,recombinant DNA constructs useful for conferring drought tolerance,compositions (such as plants or seeds) comprising these recombinant DNAconstructs, and methods utilizing these recombinant DNA constructs.

Isolated Polynucleotides and Polypeptides:

The present disclosure includes the following isolated polynucleotidesand polypeptides:

An isolated polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based onthe Clustal W method of alignment, when compared to a sequence selectedfrom the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20and 22. The polypeptide is preferably a PRE2 polypeptide.

An isolated polypeptide wherein the amino acid sequence is a sequenceselected from the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13,15, 18, 20 and 22; by alteration of one or more amino acids by at leastone method selected from the group consisting of: deletion,substitution, addition and insertion; and (c) a polypeptide wherein theamino acid sequence of the polypeptide comprises a sequence selectedfrom the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20and 22. The polypeptide is preferably a PRE2 polypeptide.

An isolated polynucleotide comprising a nucleotide sequence encoding apolypeptide with drought tolerance activity, wherein the nucleotidesequence is hybridizable under stringent conditions with a DNA moleculecomprising the full complement of a sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21,23-26; An isolated polynucleotide comprising a nucleotide sequenceencoding a polypeptide with drought tolerance activity, wherein thenucleotide sequence is a sequence selected from the group consisting ofSEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; byalteration of one or more nucleotides by at least one method selectedfrom the group consisting of: deletion, substitution, addition andinsertion.

Recombinant DNA Constructs:

In one aspect, the present disclosure includes recombinant DNAconstructs.

In one embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one regulatory sequence(e.g., a promoter functional in a plant), wherein the polynucleotidecomprises (i) a nucleic acid sequence encoding an amino acid sequence ofat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity, based on the Clustal W method of alignment, when compared to asequence selected from the group consisting of SEQ ID NOS: 3, 5, 7, 9,11, 13, 15, 18, 20 and 22; or (ii) a full complement of the nucleic acidsequence of (i).

In another embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one regulatory sequence(e.g., a promoter functional in a plant), wherein said polynucleotideencodes a PRE2 polypeptide. The PRE2 polypeptide may be from Arabidopsisthaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja andGlycine tomentella.

It is understood, as those skilled in the art will appreciate, that thedisclosure encompasses more than the specific exemplary sequences.Alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not affectthe functional properties of the encoded polypeptide, are well known inthe art. For example, a codon for the amino acid alanine, a hydrophobicamino acid, may be substituted by a codon encoding another lesshydrophobic residue, such as glycine, or a more hydrophobic residue,such as valine, leucine, or isoleucine. Similarly, changes which resultin substitution of one negatively charged residue for another, such asaspartic acid for glutamic acid, or one positively charged residue foranother, such as lysine for arginine, can also be expected to produce afunctionally equivalent product. Nucleotide changes which result inalteration of the N-terminal and C-terminal portions of the polypeptidemolecule would also not be expected to alter the activity of thepolypeptide. Each of the proposed modifications is well within theroutine skill in the art, as is determination of retention of biologicalactivity of the encoded products.

The protein of the current disclosure may also be a protein whichcomprises an amino acid sequence comprising deletion, substitution,insertion and/or addition of one or more amino acids in an amino acidsequence selected from the group consisting of SEQ ID NOS: 3, 5, 7, 9,11, 13, 15, 18, 20 and 22. The substitution may be conservative, whichmeans the replacement of a certain amino acid residue by another residuehaving similar physical and chemical characteristics. Non-limitingexamples of conservative substitution include replacement betweenaliphatic group-containing amino acid residues such as Ile, Val, Leu orAla, and replacement between polar residues such as Lys-Arg, Glu-Asp orGln-Asn replacement.

Proteins derived by amino acid deletion, substitution, insertion and/oraddition can be prepared when DNAs encoding their wild-type proteins aresubjected to, for example, well-known site-directed mutagenesis (see,e.g., Nucleic Acid Research, 10(20):6487-6500, (1982), which is herebyincorporated by reference in its entirety). As used herein, the term“one or more amino acids” is intended to mean a possible number of aminoacids which may be deleted, substituted, inserted and/or added bysite-directed mutagenesis.

Site-directed mutagenesis may be accomplished, for example, as followsusing a synthetic oligonucleotide primer that is complementary tosingle-stranded phage DNA to be mutated, except for having a specificmismatch (i.e., a desired mutation). Namely, the above syntheticoligonucleotide is used as a primer to cause synthesis of acomplementary strand by phages, and the resulting duplex DNA is thenused to transform host cells. The transformed bacterial culture isplated on agar, whereby plaques are allowed to form fromphage-containing single cells. As a result, in theory, 50% of newcolonies contain phages with the mutation as a single strand, while theremaining 50% have the original sequence. At a temperature which allowshybridization with DNA completely identical to one having the abovedesired mutation, but not with DNA having the original strand, theresulting plaques are allowed to hybridize with a synthetic probelabeled by kinase treatment. Subsequently, plaques hybridized with theprobe are picked up and cultured for collection of their DNA.

Techniques for allowing deletion, substitution, insertion and/oraddition of one or more amino acids in the amino acid sequences ofbiologically active peptides such as enzymes while retaining theiractivity include site-directed mutagenesis mentioned above, as well asother techniques such as those for treating a gene with a mutagen andthose in which a gene is selectively cleaved to remove, substitute,insert or add a selected nucleotide or nucleotides, and then ligated.Alternatively, random mutagenesis approaches may be used to disrupt or“knock-out” the expression of a Pre2 gene using either chemical orinsertional mutagenesis or irradiation. A mutagenesis and mutantidentification system known as TILLING (for targeting induced locallesions in genomes) can also be used. In this method, mutations areinduced in the seed of a plant of interest, for example, using EMStreatment. The resulting plants are grown and self-fertilized, and theprogeny are assessed. For example, the plants may be assed using PCR toidentify whether a mutated plant has a Pre2 mutation, e.g., that reducesexpression of a Pre2 gene. See, e.g., Colbert, et al., (2001) PlantPhysiol 126:480-484; McCallum, et al., (2000) Nature Biotechnology18:455-457.

The term “under stringent conditions” means that two sequences hybridizeunder moderately or highly stringent conditions. More specifically,moderately stringent conditions can be readily determined by thosehaving ordinary skill in the art, e.g., depending on the length of DNA.The basic conditions are set forth by Sambrook, et al., MolecularCloning: A Laboratory Manual, Third Edition, Chapters 6 and 7, ColdSpring Harbor Laboratory Press, 2001 and include the use of a prewashingsolution for nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH8.0), hybridization conditions of about 50% formamide, 2×SSC to 6×SSC atabout 40-50° C. (or other similar hybridization solutions, such asStark's solution, in about 50% formamide at about 42° C.) and washingconditions of, for example, about 40-60° C., 0.5-6×SSC, 0.1% SDS.Preferably, moderately stringent conditions include hybridization (andwashing) at about 50° C. and 6×SSC. Highly stringent conditions can alsobe readily determined by those skilled in the art, e.g., depending onthe length of DNA.

Generally, such conditions include hybridization and/or washing athigher temperature and/or lower salt concentration (such ashybridization at about 65° C., 6×SSC to 0.2×SSC, preferably 6×SSC, morepreferably 2×SSC, most preferably 0.2×SSC), compared to the moderatelystringent conditions. For example, highly stringent conditions mayinclude hybridization as defined above, and washing at approximately65-68° C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15 M NaCl, 10 mMNaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is0.15 M NaCl and 15 mM sodium citrate) in the hybridization and washingbuffers; washing is performed for 15 minutes after hybridization iscompleted.

It is also possible to use a commercially available hybridization kitwhich uses no radioactive substance as a probe. Specific examplesinclude hybridization with an ECL direct labeling & detection system(Amersham). Stringent conditions include, for example, hybridization at42° C. for 4 hours using the hybridization buffer included in the kit,which is supplemented with 5% (w/v) Blocking reagent and 0.5 M NaCl, andwashing twice in 0.4% SDS, 0.5×SSC at 55° C. for 20 minutes and once in2×SSC at room temperature for 5 minutes.

The protein of the present disclosure is preferably a protein withdrought tolerance activity.

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene and/or thelevel of the enzyme activity or protein functionality. The terms“suppression”, “suppressing” and “silencing”, used interchangeablyherein, include lowering, reducing, declining, decreasing, inhibiting,eliminating or preventing. “Silencing” or “gene silencing” does notspecify mechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest (e.g., Pre2) and may comprise all or part of thenucleic acid sequence of the sense strand (or antisense strand) of thetarget gene of interest. Depending upon the approach to be utilized, theregion may be 100% identical or less than 100% identical (e.g., at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to all or part ofthe sense strand (or antisense strand) of the gene of interest.

For example, an RNAi target sequence includes about 20 to about 1000contiguous bases of the disclosed Pre2 sense or anti-sense strand. In anembodiment, the target sequence includes about 50, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1100 and 1200 bases of the Pre2 sense oranti-sense strand. Within those contiguous bases, there can bevariations and the target RNAi sequences need not be identical and asdescribed above, the similarity level can range from 50% to about 99%.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs andmore generally, RNAi

(RNA interference) constructs and small RNA constructs such as siRNA(short interfering RNA) constructs and miRNA (microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target gene orgene product. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target isolated nucleic acid fragment(U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA maybe with any part of the specific gene transcript, i.e., at the 5′non-coding sequence, 3′ non-coding sequence, introns or the codingsequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target gene or geneproduct. “Sense” RNA refers to RNA transcript that includes the mRNA andcan be translated into protein within a cell or in vitro. Cosuppressionconstructs in plants have been previously designed by focusing onoverexpression of a nucleic acid sequence having homology to a nativemRNA, in the sense orientation, which results in the reduction of allRNA having homology to the overexpressed sequence (see, Vaucheret, etal., (1998) Plant J. 16:651-659 and Gura, (2000) Nature 404:804-808).

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT PublicationNumber WO 1998/36083 published on Aug. 20, 1998).

Promoter inverted repeats are also suitable to suppress the expressionof endogenous genes including Pre2. Such targeted promoter inactivationis possible by identifying the promoter region of Pre2 and constructingpromoter inverted repeat constructs.

Genome editing or genome engineering through site-directed mutagenesisby custom meganucleases with unique DNA-recognition and cleavageproperties is possible (e.g., WO 2007/047859 and WO 2009/114321). Thistechnique provides the ability to specifically modify a defined targetof interest within a genome, e.g., Pre2 genomic region. Anothersite-directed engineering is through the use of zinc finger domainrecognition coupled with the restriction properties of restrictionenzyme. See, e.g., Urnov, et al., (2010) Nat Rev Genet. 11(9):636-46;Shukla, et al., (2009) Nature 459(7245):437-41. These citations areincorporated herein to the extent they relate to materials and methodsto enable genome editing through site-specific modification. Such genomeediting techniques are used to engineer site-directed changes includingincreasing gene expression of an endogenous gene (e.g., placing anenhancer element in control of the transcription), transcriptionallysilencing an endogenous gene, creating mutants, variants of the encodedpolypeptide, removing one or more genomic regions and other methods tomodulate the gene expression and/or its activity.

Knock-out or gene knock-out refers to an inhibition or substantialsuppression of endogenous gene expression either by a transgenic or anon-transgenic approach. For example, knock-outs can be achieved by avariety of approaches including transposons, retrotransposons,deletions, substitutions, mutagenesis of the endogenous coding sequenceand/or a regulatory sequence such that the expression is substantiallysuppressed; and any other methodology that suppresses the activity ofthe target of interest.

Exogenous application of nucleotides including synthetic nucleotidemolecules to induce RNAi-mediated silencing of the endogenous Pre2 geneis possible. See e.g., US 2008/0248576, US 2011/0296556 and WO2011/112570. Exogenously applied agents are capable of inducing thedownregulation of the endogenous gene.

Regulatory Sequences:

A recombinant DNA construct of the present disclosure may comprise atleast one regulatory sequence. A regulatory sequence may be a promoter.

A number of promoters can be used in recombinant DNA constructs of thepresent disclosure. The promoters can be selected based on the desiredoutcome, and may include constitutive, tissue-specific, inducible orother promoters for expression in the host organism.

Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”.

High level, constitutive expression of the candidate gene under controlof the 35S or UBI promoter may have pleiotropic effects, althoughcandidate gene efficacy may be estimated when driven by a constitutivepromoter. Use of tissue-specific and/or stress-specific promoters mayeliminate undesirable effects but retain the ability to enhance droughttolerance. This effect has been observed in Arabidopsis (Kasuga, et al.,(1999) Nature Biotechnol. 17:287-91).

Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 1999/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171);ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611.

In choosing a promoter to use in the methods of the disclosure, it maybe desirable to use a tissue-specific or developmentally regulatedpromoter.

A tissue-specific or developmentally regulated promoter is a DNAsequence which regulates the expression of a DNA sequence selectively inthe cells/tissues of a plant critical to tassel development, seed set,or both, and limits the expression of such a DNA sequence to the periodof tassel development or seed maturation in the plant. Any identifiablepromoter may be used in the methods of the present disclosure whichcauses the desired temporal and spatial expression.

Promoters which are seed or embryo-specific and may be useful in thedisclosure include soybean Kunitz trypsin inhibitor (Kti3, Jofuku andGoldberg, (1989) Plant Cell 1:1079-1093), patatin (potato tubers)(Rocha-Sosa, et al., (1989) EMBO J. 8:23-29), convicilin, vicilin, andlegumin (pea cotyledons) (Rerie, et al., (1991) Mol. Gen. Genet.259:149-157; Newbigin, et al., (1990) Planta 180:461-470; Higgins, etal., (1988) Plant. Mol. Biol. 11:683-695), zein (maize endosperm)(Schemthaner, et al., (1988) EMBO J. 7:1249-1255), phaseolin (beancotyledon) (Segupta-Gopalan, et al., (1985) Proc. Natl. Acad. Sci.U.S.A. 82:3320-3324), phytohemagglutinin (bean cotyledon) (Voelker, etal., (1987) EMBO J. 6:3571-3577), B-conglycinin and glycinin (soybeancotyledon) (Chen, et al., (1988) EMBO J. 7:297-302), glutelin (riceendosperm), hordein (barley endosperm) (Marris, et al., (1988) PlantMol. Biol. 10:359-366), glutenin and gliadin (wheat endosperm) (Colot,et al., (1987) EMBO J. 6:3559-3564) and sporamin (sweet potato tuberousroot) (Hattori, et al., (1990) Plant Mol. Biol. 14:595-604). Promotersof seed-specific genes operably linked to heterologous coding regions inchimeric gene constructions maintain their temporal and spatialexpression pattern in transgenic plants. Such examples includeArabidopsis thaliana 2S seed storage protein gene promoter to expressenkephalin peptides in Arabidopsis and Brassica napus seeds(Vanderkerckhove, et al., (1989) Bio/Technology 7:L929-932), bean lectinand bean beta-phaseolin promoters to express luciferase (Riggs, et al.,(1989) Plant Sci. 63:47-57) and wheat glutenin promoters to expresschloramphenicol acetyl transferase (Colot, et al., (1987) EMBO J6:3559-3564).

Inducible promoters selectively express an operably linked DNA sequencein response to the presence of an endogenous or exogenous stimulus, forexample by chemical compounds (chemical inducers) or in response toenvironmental, hormonal, chemical and/or developmental signals.Inducible or regulated promoters include, for example, promotersregulated by light, heat, stress, flooding or drought, phytohormones,wounding or chemicals such as ethanol, jasmonate, salicylic acid orsafeners.

Promoters for use in the current disclosure include the following: 1)the stress-inducible RD29A promoter (Kasuga, et al., (1999) NatureBiotechnol. 17:287-91); 2) the barley promoter, B22E; expression of B22Eis specific to the pedicel in developing maize kernels (Klemsdal, etal., (1991) Mol. Gen. Genet. 228(1/2):9-16) and 3) maize promoter, Zag2(Schmidt, et al., (1993) Plant Cell 5(7):729-737; Theissen, et al.,(1995) Gene 156(2):155-166; NCBI GenBank Accession Number X80206)). Zag2transcripts can be detected 5 days prior to pollination to 7 to 8 daysafter pollination (“DAP”), and directs expression in the carpel ofdeveloping female inflorescences and Ciml which is specific to thenucleus of developing maize kernels. Ciml transcript is detected 4 to 5days before pollination to 6 to 8 DAP. Other useful promoters includeany promoter which can be derived from a gene whose expression ismaternally associated with developing female florets.

Additional promoters for regulating the expression of the nucleotidesequences of the present disclosure in plants are stalk-specificpromoters. Such stalk-specific promoters include the alfalfa S2Apromoter (GenBank Accession Number EF030816; Abrahams, et al., (1995)Plant Mol. Biol. 27:513-528) and S2B promoter (GenBank Accession NumberEF030817) and the like, herein incorporated by reference.

Promoters may be derived in their entirety from a native gene or becomposed of different elements derived from different promoters found innature or even comprise synthetic DNA segments.

Promoters for use in the current disclosure may include: RIP2, mLIP15,ZmCOR1, Rab17, CaMV 35S, RD29A, B22E, Zag2, SAM synthetase, ubiquitin,CaMV 19S, nos, Adh, sucrose synthase, R-allele, the vascular tissuepreferred promoters S2A (Genbank Accession Number EF030816) and S2B(Genbank Accession Number EF030817) and the constitutive promoter GOS2from Zea mays. Other promoters include root preferred promoters, such asthe maize NAS2 promoter, the maize Cyclo promoter (US 2006/0156439,published Jul. 13, 2006), the maize ROOTMET2 promoter (WO 2005/063998,published Jul. 14, 2005), the CR1BIO promoter (WO 2006/055487, publishedMay 26, 2006), the CRWAQ81 (WO 2005/035770, published Apr. 21, 2005) andthe maize ZRP2.47 promoter (NCBI Accession Number: U38790; GI Number1063664).

Recombinant DNA constructs of the present disclosure may also includeother regulatory sequences, including but not limited to, translationleader sequences, introns, and polyadenylation recognition sequences. Inanother embodiment of the present disclosure, a recombinant DNAconstruct of the present disclosure further comprises an enhancer orsilencer.

An intron sequence can be added to the 5′ untranslated region, theprotein-coding region or the 3′ untranslated region to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold. Buchman and Berg,(1988) Mol. Cell Biol. 8:4395-4405; Callis, et al., (1987) Genes Dev.1:1183-1200.

Any plant can be selected for the identification of regulatory sequencesand PRE2 polypeptide genes to be used in recombinant DNA constructs ofthe present disclosure. Examples of suitable plant targets for theisolation of genes and regulatory sequences would include but are notlimited to alfalfa, apple, apricot, Arabidopsis, artichoke, arugula,asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry,broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot,cassava, castorbean, cauliflower, celery, cherry, chicory, cilantro,citrus, clementines, clover, coconut, coffee, corn, cotton, cranberry,cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel,figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit,lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon,mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive,onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea,peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum,pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio,radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweetpotato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf,turnip, a vine, watermelon, wheat, yams and zucchini.

Compositions:

A composition of the present disclosure is a plant comprising in itsgenome any of the recombinant DNA constructs of the present disclosure(such as any of the constructs discussed above). Compositions alsoinclude any progeny of the plant, and any seed obtained from the plantor its progeny, wherein the progeny or seed comprises within its genomethe recombinant DNA construct. Progeny includes subsequent generationsobtained by self-pollination or out-crossing of a plant. Progeny alsoincludes hybrids and inbreds.

In hybrid seed propagated crops, mature transgenic plants can beself-pollinated to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced recombinant DNA construct.These seeds can be grown to produce plants that would exhibit an alteredagronomic characteristic (e.g., an increased agronomic characteristicoptionally under water limiting conditions), or used in a breedingprogram to produce hybrid seed, which can be grown to produce plantsthat would exhibit such an altered agronomic characteristic. The seedsmay be maize seeds.

The plant may be a monocotyledonous or dicotyledonous plant, forexample, a maize, rice or soybean plant, such as a maize hybrid plant ora maize inbred plant. The plant may also be sunflower, sorghum, canola,wheat, alfalfa, cotton, barley, millet, sugarcane, switchgrass, tobacco,potato and sugar beet.

The recombinant DNA construct may be stably integrated into the genomeof the plant.

Particularly embodiments include but are not limited to the following:

1. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence, wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity,based on the Clustal W method of alignment, when compared to a sequenceselected from the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13,15, 18, 20 and 22; and wherein said plant exhibits increased droughttolerance when compared to a control plant not comprising saidrecombinant DNA construct. The plant may further exhibit an alterationof at least one agronomic characteristic when compared to the controlplant.

2. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence, wherein saidpolynucleotide encodes a PRE2 polypeptide, and wherein said plantexhibits increased drought tolerance when compared to a control plantnot comprising said recombinant DNA construct. The plant may furtherexhibit an alteration of at least one agronomic characteristic whencompared to the control plant.

3. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence, wherein saidpolynucleotide encodes a PRE2 polypeptide, and wherein said plantexhibits an alteration of at least one agronomic characteristic whencompared to a control plant not comprising said recombinant DNAconstruct.

4. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory element, wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity,based on the Clustal W method of alignment, when compared to a sequenceselected from the group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12,14, 16, 17, 19, 21, 23-26, and wherein said plant exhibits an alterationof at least one agronomic characteristic when compared to a controlplant not comprising said recombinant DNA construct.

5. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory element, wherein saidpolynucleotide comprises a nucleotide sequence encoding a polypeptidewith drought tolerance activity, wherein the nucleotide sequence is: (a)hybridizable under stringent conditions with a DNA molecule comprisingthe full complement of a sequence selected from the group consisting ofSEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; or (b) asequence selected from the group consisting of SEQ ID NOS: 1, 2, 4, 6,8, 10, 12, 14, 16, 17, 19, 21, 23-26; by alteration of one or morenucleotides by at least one method selected from the group consistingof: deletion, substitution, addition and insertion; and wherein saidplant exhibits increased drought tolerance when compared to a controlplant not comprising said recombinant DNA construct.

6. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory element, wherein saidpolynucleotide comprises a nucleotide sequence encoding a polypeptidewith drought tolerance activity, wherein the nucleotide sequence is: (a)hybridizable under stringent conditions with a DNA molecule comprisingthe full complement of a sequence selected from the group consisting ofSEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; or (b) asequence selected from the group consisting of SEQ ID NOS: 1, 2, 4, 6,8, 10, 12, 14, 16, 17, 19, 21, 23-26; by alteration of one or morenucleotides by at least one method selected from the group consistingof: deletion, substitution, addition and insertion; and wherein saidplant exhibits an alteration of at least one agronomic characteristicwhen compared to a control plant not comprising said recombinant DNAconstruct.

8. Any progeny of the above plants in embodiments 1-7, any seeds of theabove plants in embodiments 1-7, any seeds of progeny of the aboveplants in embodiments 1-7, and cells from any of the above plants inembodiments 1-6 and progeny thereof.

In any of the foregoing embodiments 1-8 or any other embodiments of thepresent disclosure, the PRE2 polypeptide may be from Arabidopsisthaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja orGlycine tomentella.

In any of the foregoing embodiments 1-8 or any other embodiments of thepresent disclosure, the recombinant DNA construct may comprise at leasta promoter functional in a plant as a regulatory sequence.

In any of the foregoing embodiments 1-8 or any other embodiments of thepresent disclosure, the alteration of at least one agronomiccharacteristic is either an increase or decrease.

In any of the foregoing embodiments 1-8 or any other embodiments of thepresent disclosure, the at least one agronomic characteristic may beselected from the group consisting of greenness, yield, growth rate,biomass, fresh weight at maturation, dry weight at maturation, fruityield, seed yield, total plant nitrogen content, fruit nitrogen content,seed nitrogen content, nitrogen content in a vegetative tissue, totalplant free amino acid content, fruit free amino acid content, seed freeamino acid content, free amino acid content in a vegetative tissue,total plant protein content, fruit protein content, seed proteincontent, protein content in a vegetative tissue, drought tolerance,nitrogen uptake, root lodging, harvest index, stalk lodging, plantheight, ear height, ear length, salt tolerance, early seedling vigor andseedling emergence under low temperature stress. For example, thealteration of at least one agronomic characteristic may be an increasein yield, greenness or biomass.

In any of the foregoing embodiments 1-8 or any other embodiments of thepresent disclosure, the plant may exhibit the alteration of at least oneagronomic characteristic when compared, under water limiting conditions,to a control plant not comprising said recombinant DNA construct.

“Drought” refers to a decrease in water availability to a plant that,especially when prolonged, can cause damage to the plant or prevent itssuccessful growth (e.g., limiting plant growth or seed yield).

“Drought tolerance” is a trait of a plant to survive under droughtconditions over prolonged periods of time without exhibiting substantialphysiological or physical deterioration.

“Increased drought tolerance” of a plant is measured relative to areference or control plant, and is a trait of the plant to survive underdrought conditions over prolonged periods of time, without exhibitingthe same degree of physiological or physical deterioration relative tothe reference or control plant grown under similar drought conditions.Typically, when a transgenic plant comprising a recombinant DNAconstruct in its genome exhibits increased drought tolerance relative toa reference or control plant, the reference or control plant does notcomprise in its genome the recombinant DNA construct.

One of ordinary skill in the art is familiar with protocols forsimulating drought conditions and for evaluating drought tolerance ofplants that have been subjected to simulated or naturally-occurringdrought conditions. For example, one can simulate drought conditions bygiving plants less water than normally required or no water over aperiod of time, and one can evaluate drought tolerance by looking fordifferences in physiological and/or physical condition, including (butnot limited to) vigor, growth, size, or root length, or in particular,leaf color or leaf area size. Other techniques for evaluating droughttolerance include measuring chlorophyll fluorescence, photosyntheticrates and gas exchange rates.

A drought stress experiment may involve a chronic stress (i.e., slow drydown) and/or may involve two acute stresses (i.e., abrupt removal ofwater) separated by a day or two of recovery. Chronic stress may last8-10 days. Acute stress may last 3-5 days. The following variables maybe measured during drought stress and well watered treatments oftransgenic plants and relevant control plants:

The variable “% area chg_start chronic—acute2” is a measure of thepercent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and the day of thesecond acute stress

The variable “% area chg_start chronic—end chronic” is a measure of thepercent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and the last day ofchronic stress.

The variable “% area chg_start chronic—harvest” is a measure of thepercent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and the day of harvest.

The variable “% area chg_start chronic—recovery24 hr” is a measure ofthe percent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and 24 hrs into therecovery (24 hrs after acute stress 2).

The variable “psii_acute1” is a measure of Photosystem II (PSII)efficiency at the end of the first acute stress period. It provides anestimate of the efficiency at which light is absorbed by PSII antennaeand is directly related to carbon dioxide assimilation within the leaf.

The variable “psii_acute2” is a measure of Photosystem II (PSII)efficiency at the end of the second acute stress period. It provides anestimate of the efficiency at which light is absorbed by PSII antennaeand is directly related to carbon dioxide assimilation within the leaf.

The variable “fv/fm_acute1” is a measure of the optimum quantum yield(Fv/Fm) at the end of the first acute stress—(variable fluorescencedifference between the maximum and minimum fluorescence/maximumfluorescence).

The variable “fv/fm_acute2” is a measure of the optimum quantum yield(Fv/Fm) at the end of the second acute stress—(variable flourescencedifference between the maximum and minimum fluorescence/maximumfluorescence).

The variable “leaf rolling_harvest” is a measure of the ratio of topimage to side image on the day of harvest.

The variable “leaf rolling_recovery24 hr” is a measure of the ratio oftop image to side image 24 hours into the recovery.

The variable “Specific Growth Rate (SGR)” represents the change in totalplant surface area (as measured by an imaging instrument) over a singleday (Y(t)=Y0*e^(r*t)). Y(t)=Y0*e^(r*t) is equivalent to % change in Y/Δtwhere the individual terms are as follows: Y(t)=Total surface area at t;Y0=Initial total surface area (estimated); r=Specific Growth Rate day⁻¹,and t=Days After Planting (“DAP”).

The variable “shoot dry weight” is a measure of the shoot weight 96hours after being placed into a 104° C. oven.

The variable “shoot fresh weight” is a measure of the shoot weightimmediately after being cut from the plant.

The Examples below describe some representative protocols and techniquesfor simulating drought conditions and/or evaluating drought tolerance.

One can also evaluate drought tolerance by the ability of a plant tomaintain sufficient yield (at least 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% yield) in field testing under simulated ornaturally-occurring drought conditions (e.g., by measuring forsubstantially equivalent yield under drought conditions compared tonon-drought conditions, or by measuring for less yield loss underdrought conditions compared to a control or reference plant).

One of ordinary skill in the art would readily recognize a suitablecontrol or reference plant to be utilized when assessing or measuring anagronomic characteristic or phenotype of a transgenic plant in anyembodiment of the present disclosure in which a control plant isutilized (e.g., compositions or methods as described herein). Forexample, by way of non-limiting illustrations:

1. Progeny of a transformed plant which is hemizygous with respect to arecombinant DNA construct, such that the progeny are segregating intoplants either comprising or not comprising the recombinant DNAconstruct: the progeny comprising the recombinant DNA construct would betypically measured relative to the progeny not comprising therecombinant DNA construct (i.e., the progeny not comprising therecombinant DNA construct is the control or reference plant).

2. Introgression of a recombinant DNA construct into an inbred line,such as in maize, or into a variety, such as in soybean: theintrogressed line would typically be measured relative to the parentinbred or variety line (i.e., the parent inbred or variety line is thecontrol or reference plant).

3. Two hybrid lines, where the first hybrid line is produced from twoparent inbred lines and the second hybrid line is produced from the sametwo parent inbred lines except that one of the parent inbred linescontains a recombinant DNA construct: the second hybrid line wouldtypically be measured relative to the first hybrid line (i.e., the firsthybrid line is the control or reference plant).

4. A plant comprising a recombinant DNA construct: the plant may beassessed or measured relative to a control plant not comprising therecombinant DNA construct but otherwise having a comparable geneticbackground to the plant (e.g., sharing at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity of nuclear geneticmaterial compared to the plant comprising the recombinant DNA construct.There are many laboratory-based techniques available for the analysis,comparison and characterization of plant genetic backgrounds; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLP®s) and Simple SequenceRepeats (SSRs) which are also referred to as Microsatellites.

Furthermore, one of ordinary skill in the art would readily recognizethat a suitable control or reference plant to be utilized when assessingor measuring an agronomic characteristic or phenotype of a transgenicplant would not include a plant that had been previously selected, viamutagenesis or transformation, for the desired agronomic characteristicor phenotype.

Methods:

Methods include but are not limited to methods for increasing droughttolerance in a plant, methods for evaluating drought tolerance in aplant, methods for altering an agronomic characteristic in a plant,methods for determining an alteration of an agronomic characteristic ina plant, and methods for producing seed. The plant may be amonocotyledonous or dicotyledonous plant, for example, a maize, rice orsoybean plant. The plant may also be sunflower, sorghum, canola, wheat,alfalfa, cotton, barley or millet. The seed may be a maize, rice orsoybean seed, for example, a maize hybrid seed or maize inbred seed.

Methods include but are not limited to the following:

A method for transforming a cell comprising transforming a cell with anyof the isolated polynucleotides of the present disclosure. The celltransformed by this method is also included. In particular embodiments,the cell is eukaryotic cell, e.g., a yeast, insect or plant cell orprokaryotic, e.g., a bacterial cell.

A method for producing a transgenic plant comprising transforming aplant cell with any of the isolated polynucleotides or recombinant DNAconstructs of the present disclosure and regenerating a transgenic plantfrom the transformed plant cell. The disclosure is also directed to thetransgenic plant produced by this method and transgenic seed obtainedfrom this transgenic plant.

A method for isolating a polypeptide of the disclosure from a cell orculture medium of the cell, wherein the cell comprises a recombinant DNAconstruct comprising a polynucleotide of the disclosure operably linkedto at least one regulatory sequence and wherein the transformed hostcell is grown under conditions that are suitable for expression of therecombinant DNA construct.

A method of altering the level of expression of a polypeptide of thedisclosure in a host cell comprising: (a) transforming a host cell witha recombinant DNA construct of the present disclosure; and (b) growingthe transformed host cell under conditions that are suitable forexpression of the recombinant DNA construct wherein expression of therecombinant DNA construct results in production of altered levels of thepolypeptide of the disclosure in the transformed host cell.

A method of increasing drought tolerance in a plant, comprising: (a)introducing into a regenerable plant cell a recombinant DNA constructcomprising a polynucleotide operably linked to at least one regulatorysequence (for example, a promoter functional in a plant), wherein thepolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity,based on the Clustal W method of alignment, when compared to a sequenceselected from the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13,15, 18, 20 and 22; and (b) regenerating a transgenic plant from theregenerable plant cell after step (a), wherein the transgenic plantcomprises in its genome the recombinant DNA construct and exhibitsincreased drought tolerance when compared to a control plant notcomprising the recombinant DNA construct. The method may furthercomprise (c) obtaining a progeny plant derived from the transgenicplant, wherein said progeny plant comprises in its genome therecombinant DNA construct and exhibits increased drought tolerance whencompared to a control plant not comprising the recombinant DNAconstruct.

A method of increasing drought tolerance in a plant, comprising: (a)introducing into a regenerable plant cell a recombinant DNA constructcomprising a polynucleotide operably linked to at least one regulatoryelement, wherein said polynucleotide comprises a nucleotide sequenceencoding a polypeptide with drought tolerance activity, wherein thenucleotide sequence is: (a) hybridizable under stringent conditions witha DNA molecule comprising the full complement of a sequence selectedfrom the group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16,17, 19, 21, 23-26; or (b) a sequence selected from the group consistingof SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; byalteration of one or more nucleotides by at least one method selectedfrom the group consisting of: deletion, substitution, addition andinsertion; and (b) regenerating a transgenic plant from the regenerableplant cell after step (a), wherein the transgenic plant comprises in itsgenome the recombinant DNA construct and exhibits increased droughttolerance when compared to a control plant not comprising therecombinant DNA construct. The method may further comprise (c) obtaininga progeny plant derived from the transgenic plant, wherein said progenyplant comprises in its genome the recombinant DNA construct and exhibitsincreased drought tolerance when compared to a control plant notcomprising the recombinant DNA construct.

A method of evaluating drought tolerance in a plant, comprising (a)obtaining a transgenic plant, wherein the transgenic plant comprises inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence (for example, apromoter functional in a plant), wherein said polynucleotide encodes apolypeptide having an amino acid sequence of at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the ClustalW method of alignment, when compared to a sequence selected from thegroup consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20 and 22;(b) obtaining a progeny plant derived from said transgenic plant,wherein the progeny plant comprises in its genome the recombinant DNAconstruct; and (c) evaluating the progeny plant for drought tolerancecompared to a control plant not comprising the recombinant DNAconstruct.

A method of evaluating drought tolerance in a plant, comprising (a)obtaining a transgenic plant, wherein the transgenic plant comprises inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory element, wherein saidpolynucleotide comprises a nucleotide sequence encoding a polypeptidewith drought tolerance activity, wherein the nucleotide sequence is: (a)hybridizable under stringent conditions with a DNA molecule comprisingthe full complement of a sequence selected from the group consisting ofSEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; or (b) asequence selected from the group consisting of SEQ ID NOS: 1, 2, 4, 6,8, 10, 12, 14, 16, 17, 19, 21, 23-26; by alteration of one or morenucleotides by at least one method selected from the group consistingof: deletion, substitution, addition and insertion; (b) obtaining aprogeny plant derived from said transgenic plant, wherein the progenyplant comprises in its genome the recombinant DNA construct; and (c)evaluating the progeny plant for drought tolerance compared to a controlplant not comprising the recombinant DNA construct.

A method of determining an alteration of an agronomic characteristic ina plant, comprising (a) obtaining a transgenic plant, wherein thetransgenic plant comprises in its genome a recombinant DNA constructcomprising a polynucleotide operably linked to at least one regulatorysequence (for example, a promoter functional in a plant), wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity,based on the Clustal W method of alignment, when compared to a sequenceselected from the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13,15, 18, 20 and 22; (b) obtaining a progeny plant derived from saidtransgenic plant, wherein the progeny plant comprises in its genome therecombinant DNA construct; and (c) determining whether the progeny plantexhibits an alteration in at least one agronomic characteristic whencompared, optionally under water limiting conditions, to a control plantnot comprising the recombinant DNA construct.

A method of determining an alteration of an agronomic characteristic ina plant, comprising (a) obtaining a transgenic plant, wherein thetransgenic plant comprises in its genome a recombinant DNA constructcomprising a polynucleotide operably linked to at least one regulatoryelement, wherein said polynucleotide comprises a nucleotide sequenceencoding a polypeptide with drought tolerance activity, wherein thenucleotide sequence is: (a) hybridizable under stringent conditions witha DNA molecule comprising the full complement of a sequence selectedfrom the group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16,17, 19, 21, 23-26; or (b) a sequence selected from the group consistingof SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; byalteration of one or more nucleotides by at least one method selectedfrom the group consisting of: deletion, substitution, addition andinsertion; (b) obtaining a progeny plant derived from said transgenicplant, wherein the progeny plant comprises in its genome the recombinantDNA construct; and (c) determining whether the progeny plant exhibits analteration in at least one agronomic characteristic when compared,optionally under water limiting conditions, to a control plant notcomprising the recombinant DNA construct.

A method of producing seed (for example, seed that can be sold as adrought tolerant product offering) comprising any of the precedingmethods and further comprising obtaining seeds from said progeny plant,wherein said seeds comprise in their genome said recombinant DNAconstruct.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, in said introducing step said regenerable plantcell may comprise a callus cell, an embryogenic callus cell, a gameticcell, a meristematic cell or a cell of an immature embryo. Theregenerable plant cells may derive from an inbred maize plant.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, said regenerating step may comprise thefollowing: (i) culturing said transformed plant cells in a mediacomprising an embryogenic promoting hormone until callus organization isobserved; (ii) transferring said transformed plant cells of step (i) toa first media which includes a tissue organization promoting hormone;and (iii) subculturing said transformed plant cells after step (ii) ontoa second media, to allow for shoot elongation, root development or both.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, the at least one agronomic characteristic may beselected from the group consisting of greenness, yield, growth rate,biomass, fresh weight at maturation, dry weight at maturation, fruityield, seed yield, total plant nitrogen content, fruit nitrogen content,seed nitrogen content, nitrogen content in a vegetative tissue, totalplant free amino acid content, fruit free amino acid content, seed freeamino acid content, amino acid content in a vegetative tissue, totalplant protein content, fruit protein content, seed protein content,protein content in a vegetative tissue, drought tolerance, nitrogenuptake, root lodging, harvest index, stalk lodging, plant height, earheight, ear length, salt tolerance, early seedling vigor and seedlingemergence under low temperature stress. The alteration of at least oneagronomic characteristic may be an increase in yield, greenness orbiomass.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, the plant may exhibit the alteration of at leastone agronomic characteristic when compared, under water limitingconditions, to a control plant not comprising said recombinant DNAconstruct.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, alternatives exist for introducing into aregenerable plant cell a recombinant DNA construct comprising apolynucleotide operably linked to at least one regulatory sequence. Forexample, one may introduce into a regenerable plant cell a regulatorysequence (such as one or more enhancers, optionally as part of atransposable element) and then screen for an event in which theregulatory sequence is operably linked to an endogenous gene encoding apolypeptide of the instant disclosure.

Transgenic plants comprising or derived from plant cells or nativeplants with reduced Pre2 expression or activity of this disclosure canbe further enhanced with stacked traits, e.g. a crop plant having anenhanced trait resulting from expression of DNA disclosed herein incombination with herbicide tolerance and/or pest resistance traits. Forexample, plants with reduced Pre2 expression can be stacked with othertraits of agronomic interest, such as a trait providing herbicideresistance and/or insect resistance, such as using a gene from Bacillusthuringensis to provide resistance against one or more of lepidopteran,coliopteran, homopteran, hemiopteran and other insects. Known genes thatconfer tolerance to herbicides such as e.g., auxin, HPPD, glyphosate,dicamba, glufosinate, sulfonylurea, bromoxynil and norflurazonherbicides can be stacked either as a molecular stack or a breedingstack with plants expressing the traits disclosed herein. Polynucleotidemolecules encoding proteins involved in herbicide tolerance include, butare not limited to, a polynucleotide molecule encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S.Pat. Nos. 39,247; 6,566,587 and for imparting glyphosate tolerance;polynucleotide molecules encoding a glyphosate oxidoreductase (GOX)disclosed in U.S. Pat. No. 5,463,175 and a glyphosate-N-acetyltransferase (GAT) disclosed in U.S. Pat. Nos. 7,622,641; 7,462,481;7,531,339; 7,527,955; 7,709,709; 7,714,188 and 7,666,643 also forproviding glyphosate tolerance; dicamba monooxygenase disclosed in U.S.Pat. No. 7,022,896 and WO 2007/146706 A2 for providing dicambatolerance; a polynucleotide molecule encoding AAD12 disclosed in USPatent Application Publication Number 2005/731044 or WO 2007/053482 A2or encoding AAD1 disclosed in US 2011/0124503 A1 or U.S. Pat. No.7,838,733 for providing tolerance to auxin herbicides (2,4-D); apolynucleotide molecule encoding hydroxyphenylpyruvate dioxygenase(HPPD) for providing tolerance to HPPD inhibitors (e.g.,hydroxyphenylpyruvate dioxygenase) disclosed in e.g., U.S. Pat. No.7,935,869; US 2009/0055976 A1 and US 2011/0023180 A1, each publicationis herein incorporated by reference in its entirety.

Other examples of herbicide-tolerance traits that could be combined withthe traits disclosed herein include those conferred by polynucleotidesencoding an exogenous phosphinothricin acetyltransferase, as describedin U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675;5,561,236; 5,648,477; 5,646,024; 6,177,616 and 5,879,903. Plantscontaining an exogenous phosphinothricin acetyltransferase can exhibitimproved tolerance to glufosinate herbicides, which inhibit the enzymeglutamine synthase. Other examples of herbicide-tolerance traits includethose conferred by polynucleotides conferring altered protoporphyrinogenoxidase (protox) activity, as described in U.S. Pat. Nos. 6,288,306 B1;6,282,837 B1 and 5,767,373 and International Patent Publication WO2001/12825. Plants containing such polynucleotides can exhibit improvedtolerance to any of a variety of herbicides which target the protoxenzyme (also referred to as “protox inhibitors”).

The introduction of recombinant DNA constructs of the present disclosureinto plants may be carried out by any suitable technique, including butnot limited to direct DNA uptake, chemical treatment, electroporation,microinjection, cell fusion, infection, vector-mediated DNA transfer,bombardment or Agrobacterium-mediated transformation. Techniques forplant transformation and regeneration have been described inInternational Patent Publication WO 2009/006276, the contents of whichare herein incorporated by reference.

The development or regeneration of plants containing the foreign,exogenous isolated nucleic acid fragment that encodes a protein ofinterest is well known in the art. The regenerated plants may beself-pollinated to provide homozygous transgenic plants. Otherwise,pollen obtained from the regenerated plants is crossed to seed-grownplants of agronomically important lines. Conversely, pollen from plantsof these important lines is used to pollinate regenerated plants. Atransgenic plant of the present disclosure containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

TABLE 1A Expression of maize Pre2 in different tissues as compiled fromMPSS-Signature Platform Expression Tissue (PPTM) Dev. Stage TreatmentLeaf 2880 V5 ECB Kernel 1750 R1 Drought Stress Anther 1270 VT ControlApical Meristem, pre-floral 1110 V3 Endosperm 1080 R1 In vitro ImmatureEar 800 V9 Pedicel and Basal Layer 710 R3 Root 560 V12 HydroponicLateral Branch Meristem 540 V8 Pericarp 460 R4 Stalk 390 VnColletotrichum Leaf midrib 370 V7 Nitrate 4 h Stalk internode 350V10-V11 meristematic zone Germination Embryo 320 VE Root cortex 320 V1Nitrate-4 hr Aleurone 280 R3 Stalk nodal plate 270 V10-V11 VegetativeLateral 200 V8 Meristems Stalk rind 170 V10-V11 Root stele 100 V1Nitrate-4 hr Germination Scutellum 90 VE Tassel Spikelet 90 VT TiltHerbicide Pollen 70 VT Silk 30 R1

TABLE 1B Expression of maize Pre2 in different tissues as compiled fromMPSS-Classic Platform Tissue Expression (PPTM) Dev. Stage TreatmentEmbryo 990 R2 Aerial Vegetative 950 Vn Apical Meristem 820 Vn Root 770V6-V8 Stalk 760 V6 Immature ear 760 Vn Stalk Node 580 V12-V13 Ear Shoot530 V11 Leaf 500 V6-V8 Transgene Pericarp 490 R4 Stalk Internode Rind440 V12-V13 Leaf-base 390 V3 Leaf Whorl ECB 390 V5 ECB infestationEndosperm 380 R5 Stalk Internode 340 VT Pedicel 340 R1-R2 Drought stressKernel 340 R2 Tassel Spikelet 320 VT Root Tip Meristem 300 V6 Ovary 290Vn Silk 290 VT Tassel 280 Vn Pollen 280 VT Stem, Sheath 260 V7-V8 Ear230 V15-R1 Stalk Internode Pith 220 VT Mesocotyl 110 VE Stalk LeafPulvinus 100 VT Husk 80 R1 Stalk Node 40 V12-V13

TABLE 1C Expression of maize Pre2 in different tissues as compiled fromSolexa-WgT Platform Tissue Expression (PPTM) Dev. Stage Tassel 359.04 V6Root 349.75 V19 Immature Ear 319.93 V8 Embryo 270.21 VE Leaf 230.42 V19Kernel 211.82 R2 Root Hair 188.91 V1 Endosperm 173.4 R4 Pericarp 137.11R4 Stalk 94.79 V8 Pollen 52.79 R1

EXAMPLES

The Examples described below form part of the detailed description ofthe disclosure. The present disclosure is further illustrated in thefollowing Examples, in which parts and percentages are by weight anddegrees are Celsius, unless otherwise stated. It should be understoodthat these Examples, while indicating preferred embodiments of thedisclosure, are given by way of illustration only. From the abovediscussion and these Examples, one skilled in the art can ascertain theessential characteristics of this disclosure, and without departing fromthe spirit and scope thereof, can make various changes and modificationsof the disclosure to adapt it to various usages and conditions. Thus,various modifications of the disclosure in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

Example 1 Characterization of the Pre-Mature Senescence2 (Pre2) Mutationin Maize

Forward genetics was used to clone a pre-mature senescence2 (pre2)mutation isolated from a highly Mu-active stock. The senescing phenotypeof pre2, which inherits in a recessive manner, is apparent 2-3 weeksprior to anthesis. Like natural senescence, the pre2 phenotype startsfrom the lowermost leaves and then spreads to the top of the plant in aprogressive fashion (FIG. 1A). We have cloned a candidate gene for pre2mutation using SAIFF protocol (Selective Amplification of InsertionFlanking Fragments). The candidate gene co-segregates completely withthe phenotype in a population of 500 segregating F2 plants (FIG. 1B).The pre2 encodes a conserved protein of no previously known function andis expressed at very low level (less than 100 PPM) in almost all partsof corn plant. The pre2 mutant phenotype was found to be the result ofan interference of the insertion in differential splicing of intron1 inthe transcript (FIG. 2A), which further leads to an early terminationcodon in its peptide. The Mu insertion in the mutant resulted inexpression 4 different species of mRNA with variable expression levels(FIG. 2A). In addition to wild type mRNA, the mutant also expressesmRNAs with 122, 170 and 373 bp insertions which due to pre mature stopcodons translated into predicted polypeptides of 113, 49 and 113 aminoacid residues in addition to 1271 amino acid wild type polypeptide (FIG.2A). Reverse genetics and allelic test of two independent mutant alleles(pre2-2 and pre2-3) provided proof-of-validation that the right gene forpre2 mutation had been cloned (FIG. 2C). Only a few partial ESTsrepresenting 3′ end of the gene were found in the database, thus a fulllength cDNA of 3.9 kb was amplified using RT-PCR (FIG. 2B) and clonedinto a cloning vector. The maize pre2 gene includes of 13 exons and 12introns and has 1271 amino acid long peptide. The Zmpre2 gene was mappedto chr4 on bin 189 cM. The maize Pre2 gene expression was compiled fromdifferent libraries developed by DuPont-Pioneer using various corntissues at different developmental stages under different treatments.The gene expression value measured in PPTM by using three platforms,MPSS-Signature, MPSS-Classic and Solexa-WgT, is summarized in Table 1A,1B, and 10. The Pre2 gene expression is enhanced under drought stress,insect infestation, disease inoculation, herbicide spray, and Nitrateapplication. The Pre2 is expressing in almost all plant parts of cornwith maximum expression in leaf at V5 stage followed by kernel, anther,embryo, apical meristem, and root at V6-V8 stage.

Example 2 Identification and Characterization of the Pre2 Knock-OutMutant in Arabidopsis

Homologous sequence of Pre2 in Arabidopsis was identified by using corncandidate gene sequence for pre-mature senescence2 as query. Then byusing Atpre2 gene sequence, three independent T-DNA insertional alleles(Salk_017615, Salk_079273, Salk_107247) were identified in theArabidopsis T-DNA mutant database. As in both SALK_079273 andSALK_107247 lines the T-DNA is situated in the 3′ UTR region of thecandidate gene (FIG. 4; top panel), Salk_017615 was analyzed in whichthe T-DNA is present in the coding sequence. This mutant line wasobtained from ABRC and plants were grown and subjected to PCRfingerprinting and RT-PCR analyses. PCR amplification of the T-DNAflanking sequences using gene specific primer along with T-DNA primerconfirmed that the T-DNA insertion is present in exon10 of At-PRE2 gene(FIG. 4; upper panel). Genomic PCRs using gene and T-DNA specificprimers also showed that all plants were having T-DNA in the Atpre2 gene(FIG. 3, 2^(nd) panel from top). The gene specific primers flanking theT-DNA insertion will amplify DNA region in wild-type (WT) plants andright size PCR product was present in all plant except plant #11 and #25(FIG. 3, 3^(rd) panel from top) indicating that all plants except #11and #25 were heterozygous for this insertion. PCR amplification of Actinin both mutant and wild type plants was used as control (FIG. 4; 4^(th)panel from top). Based on these genotyping results plant #11 and 25 wereidentified as homozygous for T-DNA insertion. Expression of Pre2 is lowin Arabidopsis and almost present in plant parts but highest was noticedin siliques and maturing seeds (FIG. 3). Furthermore, ReverseTranscriptase Polymerase Chain Reaction (RT-PCR) was performed on theseplants and, but a full length transcript of AtPre2 mRNA was not detectedin plant #11 and #25 (35 cycles) indicating that AtPre2 gene expressionis knocked out in these two T-DNA mutants. We harvested seed from thetwo homozygous and all heterozygous plants. In order to identify andmultiply the seed of WT-sib (+/+), seeds from the next generation from aself progeny of heterozygous plant were grown and PCR fingerprinting wasrepeated. The homozygous nature of T-DNA knock out in plant #11 and #25was confirmed and the seeds were multiplied. Morphological traits fromboth homozygous plant #11 and #25 were compared with homozygous WT-sib(+/+) and heterozygous WT-sib (+/Pre2) at flowering. Both homozygousmutants were robust in growth with more pod numbers but were late inmaturity by 4 to 5 days as compared to its WT-sibs (FIG. 5A). Formeasuring total biomass, 9 whole plants, each of knock out #11, knockout #25, homozygous WT, and heterozygous WT-sibs, were harvested and airdried for 14 days at room temperature. Total weight was determined byweighing and average and standard deviation were calculated forstatistical analysis. The total biomass of both knockouts (combined) wasfound to be significantly higher (t test at P<0.01) when compared toboth homozygous and heterozygous WT-sibs (FIG. 5B).

Example 3 Overexpression of Atpre2 in Arabidopsis

Multisite Gateway (Invitrogen) technology was used to generate plantexpression vectors. A 3978 bp coding sequence of AtPre2 (at1g72390) wasamplified by PCR using forward and reverse gene specific primers(GSP-F+GSP-R) and cloned in pENTR.D.TOPO. The final expression vector(pRG1261) contained herbicide and fluorescent marker for transgenic seedsorting. Quality check was performed on the resulting expression vectorby restriction digestion mapping and transferred into Agrobacteriumtumefaciens LB4404JT by electroporation. The co-integrated DNA fromtransformed Agrobacterium was transferred in E. coli DH10B and theplasmid DNA from this strain was used to check its quality again byrestriction digestion. These overexpression vectors were transformed into Arabidopsis thaliana ecotype Columbia-0 by Agobacterium mediatedFloral-Dip′ method (Clough and Bent, (1998) Plant Journal 16:735). T₀seeds were screened for T1 transformants in soil for herbicideresistance. For molecular analysis of the transgenic T1 events, RT-PCRswere conducted to detect the transgene expression, actin control and thepresence of genomic DNA in the RNA preparations. Transgene expressingevents were advanced for further studies. Overrexpression of ZmPre2coding sequence in Arabidopsis resulted in a hypersensitive response todrought. (See, FIG. 6B).

Example 4 Sub-Cellular Localization and Regulation of Expression ofAtpre2

In order to determine the sub-cellular localization AcGFP was fused inthe c-terminal of AtPRE2. This fusion cassette was either driven by 35Spromoter (pRG1263) or by ATPRE2 promoter (518 bp region upstream ofstart codon of Atpre2) in pRG1264. Similarly, in order to study theregulation of expression of AtPre2 in details, this 518 bp promoterregion of Atpre2 was fused to GUS:RFP (a dual reporter) to generatepRG1265. All these constructs were transformed into Arabidopsis asdescribed in Example 3.

Example 5 Drought Analysis of T-DNA Knockout Mutant and Over-ExpressedPre2 in Arabidopsis

Drought assay was performed on total 72 mutants and 72 wild-type sibs(WT) by using 8 pots (cells) for each. Each pot was sown to produce 9mutant s or WT seedlings in a 3×3 array. Flats are configured with 8square pots each in one experiment. Each pot was filled with Scotts®Metro-Mix® 200 soil. The soil was watered to saturation and then plantswere grown under standard conditions of 16 hour light, 8 hour darkcycle; 22° C.; “60% relative humidity). No additional water was givenafter day 16^(th).

Digital images of the plants were taken at the onset of visible droughtstress symptoms. Images were taken once a day (at the same time of day),until the plants appear dessicated. Typically, four consecutive days ofdata is captured. Color analysis was employed for identifying potentialdrought tolerant lines. Color analysis can be used to measure theincrease in the percentage of leaf area that falls into a yellow colorbin. Using hue, saturation and intensity data (“HSI”), the yellow colorbin consists of hues 35 to 45. Maintenance of leaf area was also used asanother criterion for identifying potential drought tolerant lines,since Arabidopsis leaves wilt during drought stress. Maintenance of leafarea can be measured as reduction of rosette leaf area over time. Leafarea was measured in terms of the number of green pixels obtained usingan imaging system. Mutant and control (e.g. wild-type) plants were grownside by side in flats and when wilting begins. From these data wiltingprofiles are determined based on the green pixel counts obtained overfour consecutive days for activation-tagged or knockout mutant plantsand accompanying control plants. The profile was selected from a seriesof measurements over the four day period that provided the largestdegree of wilting. The ability to withstand drought was measured by thetendency of plants to resist wilting compared to control their WT-sibplants (FIG. 6A).

Software was used to analyze CCD images. Estimates of the leaf area ofthe Arabidopsis plants were obtained in terms of the number of greenpixels. The data for each image was averaged to obtain estimates of meanand standard deviation for the green pixel counts for activation-taggedand wild-type plants. Parameters for a noise function were obtained bystraight line regression of the squared deviation versus the mean pixelcount using data for all images in a batch. Error estimates for the meanpixel count data were calculated using the fit parameters for the noisefunction. The mean pixel counts for activation-tagged and wild-typeplants are summed to obtain an assessment of the overall leaf area foreach image. The four-day interval with maximal wilting was obtained byselecting the interval that corresponds to the maximum difference inplant growth. The individual wilting responses of the activation-tagged,knockout mutants, and wild-type plants were obtained by normalization ofthe data using the value of the green pixel count of the first day inthe interval. The drought tolerance of the activation-tagged or komutant plants compared to the wild-type plant was scored by summing theweighted difference between the wilting response of mutant oractivation-tagged plants and wild-type plants over day two to day four;the weights were estimated by propagating the error in the data. Apositive drought tolerance score corresponds to an activation-tagged ormutant plant with slower wilting compared to the wild-type plant.Significance of the difference in wilting response betweenactivation-tagged and wild-type plants was obtained from the weightedsum of the squared deviations.

In drought assay the Atpre2 mutant plants were showing positive scoregreater than 0.9 with positive standard deviation in all flats. Thisdemonstrated that these mutant plants outperformed significantly betterthan their wild type sibs used as control (FIG. 6B). The second controlused in this experiment was ZmPre2 gene over expressed under 35Spromoter in Arabidopsis. These plants became hypersensitive to droughtstress (FIG. 6B) further authenticated these drought assay results.

Example 6 Analysis of Atpre2 Mutants on Low and High Nitrogen

For low nitrogen (Low N) plate assays, 32 mutant and 32 wild type plantswere grown on square plates (15 mm×15 mm) containing 0.5×N-FreeHoagland's, 0.4 mM potassium nitrate, 0.1% sucrose, 1 mM MES and 0.25%Phytagel™ (Low N medium). Plates were kept for three days in the dark at4° C. to stratify seeds and then placed horizontally for nine days at22° C. light and 20° C. dark. Plates were placed under sixteen hourslight and eight hours dark, with an average light intensity of ˜200mmol/m²/s. Plates were rotated and shuffled daily within each shelf. Atday twelve (nine days of growth), seedling status was evaluated byimaging the entire plate. After masking the plate image to removebackground color, two different measurements were collected for eachindividual plant: total rosette area, and the percentage of color thatfalls into a green color bin using hue, saturation and intensity data(HSI). The green color bin consists of hues 50 to 66. Total rosette areawas used as a measure of plant biomass, whereas the green color bin wasshown by dose-response studies to be an indicator of nitrogenassimilation. In this assay Atpre2 mutant plants showed a significantlyhigher total area (biomass) and green color (Bin2 area) (FIG. 7).

For high nitrogen (High N) root assays, 16 mutants and 16 wild typeplants instead of 32 each were grown on plates in the same light andtemperature conditions as described above. The plates were having thesame medium except it was containing 60 mM of potassium nitrate. N androot biomass was measured by imaging. Four independent experiments wereperformed and the data revealed that in each case mutant plants werehyper-sensitive to higher concentration of nitrogen which leads tosevere root growth inhibition as compared to its wild type sib plants(FIG. 8).

Example 7 Down-Regulation of Endogenous ZmPre2 mRNA by RNAi Studies

A genomic fragment of 450 bp (from 1189 to 1638nt of ZmPre2 CDS) wasused in sense and antisense orientation with an intron (ST-SL2 intron2)as a spacer to make an inverted repeat/RNAi cassette. This cassette wasdriven by either Zm-UBI (constitutive promoter) and/or a putativeZM-SEE1 (senescence induced promoter) promoters. MOPAT driven by Zm-UBIpromoter and PMI driven by OsACTIN promoter was used as selectablemarkers. In addition, RFP driven by a pericarp specific promoter LTP2was also used to sort out the transgenic seeds (red) from theirsegregating non-transgenic sib seeds. Transgenic lines for theconstructs were generated and molecular analyses, such as PCR-FP andRT-PCR, were performed for selection of transgenic events. Several lineswith significantly reduced expression of ZmPre2 have been identified andare characterized in further experiments.

Example 8 Down-Regulation of Endogenous ZmPre2-mRNA by RNAi Studies

ZmPre2 RNAi suppression construct is transformed into a fast cyclingcorn line (FASTCORN) for further transgenic validation. A full lengthmRNA amplified by RT-PCR (FIG. 2A) was used to make both forover-expression (Ox) and RNAi constructs using Ubi promoter. Tentransgenic events for both were screened molecularly for copy number andPre2 gene expression by QPCR. For phenotypic data on leaf area, leafcolor, height etc. digital images of the plants at various growth stageswere taken as described above. Data on total biomass and stay greentraits were calculated by measuring the leaf area in terms of the numberof green pixels obtained using a commercially available imaging system.Data for other traits such as ear length, ear width, maximum ear areaand total seed number were obtained at the time of harvesting. Data wasanalyzed by applying paired t-test and presented as Z-score in FIG. 9.All ten events (RNAi construct) and all but two of the (Ox) had singlecopy and the relative gene expression in five out ten RNAi events wassignificantly low (ranging from 0.07 to 0.554) as compared to internaltransgenic and non-transgenic controls. All but one Ox events had 2×more relative expression ranging from 2.171 to 2.897. Three RNAi events(1.4, 1.5 and 2.5) were found to have significantly higher ear length,ear width and total seed numbers (FIG. 9), which is relevant to theirrelative gene expression. However, pre-mature senescence phenotype wasnot observed in these events. This could be due to the fact that all theinsertion mutant alleles for the native Pre2 gene were resulting fromthe partial interference and differential splicing of the introns intheir mature transcript, whereas the RNAi mechanism is different thanMutator insertion mutants. On the other hand, these four RNAi eventswith higher seed numbers as compared to all overexpression (Ox) eventsare behaving similar to T-DNA knockouts in Arabidopsis. These fourevents also show higher biomass (FIG. 9A). Three RNAi events (namely1.4, 1.5 and 2.5) were selected for conducting NUE Reproductive Assay inT1 generation under 4.0 mMol Nitrate-suboptimal nitrogen conditions. Twoof three events (1.5 and 2.5) showed significant increase (percentchange vs. Null) in silk count, ear length, ear width and ear area (FIG.9B). In addition to these traits, event 2.5 also showed significantdifference for Days to shed and days to silk as compared to its nulls.Thus, transgenic plants where the expression of the Pre2 mRNA has beenmodulated exhibit significant differences in one or more agronomicparameters of interest for crop plants.

Example 9 Characterization of Polypeptides Homologous to Pre2

The protein-coding regions of other genes homologous to the PRE2 aminoacid sequences disclosed herein. FIGS. 10-11 present an alignment of aplurality of amino acid sequences set forth in SEQ ID NOS: 1-48.

Sequence alignments and percent identity calculations were performedusing the MEGALIGN® program of the LASERGENE® bioinformatics computingsuite (DNASTAR® Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal W method of alignment (Higginsand Sharp (1989) CAB/OS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

The amino acid sequence of corn Pre2 peptide (ZmPRE2) has the followingpercent sequence identity with the homologs presented in FIGS. 10 and11: Pre2 peptides of sorghum and grasses such as Sudan, Bahia andResurrection were found to be 83% to 90% identical with corn at aminoacid level whereas the rice peptide diverged from corn and showed 68%.Homologs in dicots including Arabidopsis, Soybean and Canola have 34%,36%, and 35% identity, respectively at the global alignment level.

Example 10 Molecular Analysis of the PRE2 Homologs

Molecular analysis revealed several conserved regions/domain in the Pre2homologs. Despite the overall sequence divergence along the full-lengthof the Pre2 polypeptides across a variety of species shown in FIG. 10for example, several highly conserved domains were observed (FIG. 11).SEQ ID NOS: 27-48 represent a subset of conserved regions and domainsacross the Pre2 polypeptide region.

Example 11 Expression of Transgenes or Downregulation of EndogenousGenes in Soybean

Local Blast results using AtPre2 full length gene sequence as queryshowed that there are two copies of Pre2 gene in soybean and theirpartial sequences is aligned in a multiple alignment (FIG. 10). Apartial EST sequence (PSO423639) of about 2800 bp in length was cloned.The expression pattern distribution of the ESTs or full-length cDNAs inthe Tissue Library Browser indicate that Pre2 gene expression is verylow in soybean and Pre2-ESTs have been expressed highest in seedling,mostly in shoot under biotic and abiotic stresses. Medium numbers ofESTs (20-30) have been detected in leaf, root, young cotyledons, lowlevels in reproductive tissues such as pod, seed and seed coat.

Sequences of both the partial Pre2 copies in soybean were aligned andthe following consensus sequence of 147 bp (SEQ ID NO: 51) was selectedto use in an RNAi construct using Arabidopsis UBI promoter. Thisconstruct is transformed into soybean.

Soybean embryos are bombarded with a plasmid comprising a preferredpromoter operably linked to a heterologous nucleotide sequencecomprising a suitable target RNAi sequence against Pre2 polynucleotidesequence or subsequence (e.g., SEQ ID NOS: 14, 16, 17 and 19), asfollows. To induce somatic embryos, cotyledons of 3 to 5 mm in lengthare dissected from surface-sterilized, immature seeds of the soybeancultivar A2872, then cultured in the light or dark at 26° C. on anappropriate agar medium for six to ten weeks. Somatic embryos producingsecondary embryos are then excised and placed into a suitable liquidmedium. After repeated selection for clusters of somatic embryos thatmultiply as early, globular-staged embryos, the suspensions aremaintained as described below.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with fluorescentlights on a 16:8 hour day/night schedule. Cultures are sub-culturedevery two weeks by inoculating approximately 35 mg of tissue into 35 mlof liquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette of interest, comprising thepreferred promoter and a heterologous Pre2 polynucleotide e.g., in thesense or anti-sense or hairpin orientation, can be isolated as arestriction fragment. This fragment can then be inserted into a uniquerestriction site of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M) and 50 μl CaCl2(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×5 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 12 Transformation of Maize Using Agrobacterium

Agrobacterium-mediated transformation of maize is performed for example,as described by Zhao, et al., (2006) Meth. Mol. Biol. 318:315-323 (seealso, Zhao, et al., (2001) Mol. Breed. 8:323-333 and U.S. Pat. No.5,981,840 issued Nov. 9, 1999, incorporated herein by reference). Thetransformation process involves bacterium innoculation, co-cultivation,resting, selection and plant regeneration.

1. Immature Embryo Preparation:

Immature maize embryos are dissected from caryopses and placed in a 2 mLmicrotube containing 2 mL PHI-A medium.

2. Agrobacterium Infection and Co-Cultivation of Immature Embryos:

2.1 Infection Step:

PHI-A medium of (1) is removed with 1 mL micropipettor, and 1 mL ofAgrobacterium suspension is added. The tube is gently inverted to mix.The mixture is incubated for 5 min at room temperature.

2.2 Co-culture Step:

The Agrobacterium suspension is removed from the infection step with a 1mL micropipettor. Using a sterile spatula the embryos are scraped fromthe tube and transferred to a plate of PHI-B medium in a 100×15 mm Petridish. The embryos are oriented with the embryonic axis down on thesurface of the medium. Plates with the embryos are cultured at 20° C.,in darkness, for three days. L-Cysteine can be used in theco-cultivation phase. With the standard binary vector, theco-cultivation medium supplied with 100-400 mg/L L-cysteine is criticalfor recovering stable transgenic events.

3. Selection of Putative Transgenic Events:

To each plate of PHI-D medium in a 100×15 mm Petri dish, 10 embryos aretransferred, maintaining orientation and the dishes are sealed withPARAFILM®. The plates are incubated in darkness at 28° C. Activelygrowing putative events, as pale yellow embryonic tissue, are expectedto be visible in six to to eight weeks. Embryos that produce no eventsmay be brown and necrotic, and little friable tissue growth is evident.Putative transgenic embryonic tissue is subcultured to fresh PHI-Dplates at two-three week intervals, depending on growth rate. The eventsare recorded.

4. Regeneration of T0 plants:

Embryonic tissue propagated on PHI-D medium is subcultured to PHI-Emedium (somatic embryo maturation medium), in 100×25 mm Petri dishes andincubated at 28° C., in darkness, until somatic embryos mature, forabout ten to eighteen days. Individual, matured somatic embryos withwell-defined scutellum and coleoptile are transferred to PHI-F embryogermination medium and incubated at 28° C. in the light (about 80 μEfrom cool white or equivalent fluorescent lamps). In seven to ten days,regenerated plants, about 10 cm tall, are potted in horticultural mixand hardened-off using standard horticultural methods.

Media for Plant Transformation:

-   -   1. PHI-A: 4 g/L CHU basal salts, 1.0 mL/L 1000× Eriksson's        vitamin mix, 0.5 mg/L thiamin HCl, 1.5 mg/L 2,4-D, 0.69 g/L        L-proline, 68.5 g/L sucrose, 36 g/L glucose, pH 5.2. Add 100 μM        acetosyringone (filter-sterilized).    -   2. PHI-B: PHI-A without glucose, increase 2,4-D to 2 mg/L,        reduce sucrose to 30 g/L and supplement with 0.85 mg/L silver        nitrate (filter-sterilized), 3.0 g/L GELRITE®, 100 μM        acetosyringone (filter-sterilized), pH 5.8.    -   3. PHI-C: PHI-B without GELRITE® and acetosyringonee, reduce        2,4-D to 1.5 mg/L and supplemente with 8.0 g/L agar, 0.5 g/L        2-[N-morpholino]ethane-sulfonic acid (MES) buffer, 100 mg/L        carbenicillin (filter-sterilized).    -   4. PHI-D: PHI-C supplemented with 3 mg/L bialaphos        (filter-sterilized).    -   5. PHI-E: 4.3 g/L of Murashige and Skoog (MS) salts, (Gibco, BRL        11117-074), 0.5 mg/L nicotinic acid, 0.1 mg/L thiamine HCl, 0.5        mg/L pyridoxine HCl, 2.0 mg/L glycine, 0.1 g/L myo-inositol, 0.5        mg/L zeatin (Sigma, Cat. No. Z-0164), 1 mg/L indole acetic acid        (IAA), 26.4 μg/L abscisic acid (ABA), 60 g/L sucrose, 3 mg/L        bialaphos (filter-sterilized), 100 mg/L carbenicillin        (filter-sterilized), 8 g/L agar, pH 5.6.    -   6. PHI-F: PHI-E without zeatin, IAA, ABA; reduce sucrose to 40        g/L; replacing agar with 1.5 g/L GELRITE®; pH 5.6.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm, et al., (1990) Bio/Technology 8:833-839).

Transgenic T0 plants can be regenerated and their phenotype determined.T1 seed can be collected. T1 plants, and/or their progeny, can be grownand their phenotype determined.

Example 13 Transformation of Brassica with Pre2 Homologs DisclosedHerein

Canola transformation is accomplished for example, as described in Chenand Tulsieram, US Patent Application Publication Number 2007/0107077,incorporated herein by reference. Buds are collected from a donor lineand sterilized. Buds are then homogenized, filtered, and washed tocollect the microspores. The resultant microspore suspension wasadjusted to a specified density and cultured for 2 days. Embryogenicmicrospores were then isolated via gradient centrifugation and cultured.

Gold particles coated with the DNA fragment were used fortransformation. Biolistic transformation is carried out using thePDS-1000/He Particle Delivery System (Bio-Rad, Hercules, Calif.) asdescribed by Klein, et al., (1987) Nature 327:70-73. Transformedembryogenic microspores are cultured in fresh medium in dark conditionsfor 10-12 days, then under dim light for 1-3 weeks. Green embryos aretransferred to fresh medium and cultured for two weeks to select basedon the marker gene used. Germinated shoots and/or plants weretransferred to growth medium supplemented with selection component.

Example 14 Yield Analysis of Plants Transformed with Pre2 TargetingConstructs

A recombinant DNA construct containing a Pre2 down-regulating constructcan be introduced into plants either by direct transformation orintrogression from a separately transformed line.

Transgenic plants, either inbred or hybrid, can undergo more vigorousfield-based experiments to study yield enhancement and/or stabilityunder well-watered and water-limiting conditions.

Subsequent yield analysis can be done to determine whether plants thatcontain the constructs/sequences disclosed herein have an improvement inyield performance under water-limiting conditions, when compared to thecontrol plants that do not contain the validated drought tolerant leadgene. Specifically, drought conditions can be imposed during theflowering and/or grain fill period for plants that contain theconstructs/sequences disclosed herein and the control plants. Reductionin yield can be measured for both. Plants containing theconstructs/sequences disclosed herein have less yield loss relative tothe control plants, for example, at least 25% less yield loss, underwater limiting conditions, or would have increased yield relative to thecontrol plants under water non-limiting conditions.

The above method may be used to select transgenic plants with increasedyield, under water-limiting conditions and/or well-watered conditions,when compared to a control plant not comprising said recombinant DNAconstruct.

Example 15 At-Pre2 Mutant is Hypersensitive to ABA

In earlier experiments At-Pre2 T-DNA knock out mutant showed asignificant increase in biomass, improved growth on low N plates, anddrought tolerant phenotype in soil. AT-PRE2 is a large protein of 1326amino acid residues with unknown function. To elucidate the function ofthis protein, several experiments were conducted. One of suchexperiments included ABA response of Atpre2 mutant. Seeds of Atpre2mutant and Col-0 WT (36 seeds of each WT and mutant with 3 replications)were grown on half MS media (without sucrose) with or without abscisicacid (1 μM±-cis, trans-ABA). The plates with seeds were kept at 4° C. indark for 3 days and then incubated in growth chamber under the long daygrowth conditions (16-h-light/8-h-dark cycle at 120-150 μmol m-2 sec-1and 20° C. to 22° C., with 75% humidity). Visible radicle tips (1-2 mm)were counted after 48 hrs as a germinated seed. In these multipleexperiments Atpre2 mutant showed a hypersensitive response to ABA in adosage dependent manner. The seed germination in mutant was reduced ordelayed by more than 50% as compare to wild type in presence of 1 μM ABA(FIG. 12). Searches of expression databases revealed that the endogenousAT-PRE2 gene expression was higher in guard cells in wild type plantsand was down-regulated by ABA treatment both in seedling and leaf. Inaddition AtPRE2 was also up-regulated by nitrate in roots. These resultsindicate a direct or indirect role of AtPRE2 in ABA and Nsignaling/pathway.

1. A method of altering an agronomic parameter of a plant, the methodcomprising downregulating the endogenous expression of a nucleotideencoding a polypeptide, wherein the polypeptide comprises a conserveddomain selected from the group consisting of SEQ ID NOS: 27-48.
 2. Themethod of claim 1, wherein the agronomic parameter is selected from thegroup consisting of greenness, yield, growth rate, biomass, plantnitrogen content, drought tolerance, nitrogen uptake, root lodging,harvest index, stalk lodging, plant height, ear height, ear width, earlength, ear area, salt tolerance, early seedling vigor and seedlingemergence under low temperature stress, drought tolerance, increasednitrogen use efficiency, silk count, inducing early maturity, delayingmaturity, days to shed and days to silk.
 3. The method of claim 1,wherein the suppression of endogenous expression of the messenger RNA isby RNAi.
 4. The method of claim 1, wherein the polypeptide comprises theamino acid sequence of SEQ ID NO: 3 or a sequence that is at least 90%identical to SEQ ID NO:
 3. 5. A plant comprising in its genome apolynucleotide operably linked to at least one heterologous regulatoryelement, wherein said polynucleotide comprises a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding a polypeptide with drought tolerance activity, wherein thepolypeptide has an amino acid sequence of at least 90% sequenceidentity, based on the Clustal W method of alignment with pairwisealignment default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=0.2,DELAY DEVERGENT SEQS(%)=30%, DNA TRANSITION WEIGHT=0.5, PROTEIN WEIGHTMATRIX “Gonnet Series”), when compared to a sequence selected from thegroup consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20 and 22 ora fragment thereof; (b) a nucleotide sequence encoding a polypeptidewith drought tolerance activity, wherein the nucleotide sequence ishybridizable under stringent conditions with a DNA molecule comprisingthe full complement compared to a sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21,23-26; (c) a nucleotide sequence encoding a polypeptide with droughttolerance activity, wherein the nucleotide sequence is derived from oneor more SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26; byalteration of one or more nucleotides by at least one method selectedfrom the group consisting of: deletion, substitution, addition andinsertion; (d) a nucleotide sequence encoding a polypeptide wherein theamino acid sequence of the polypeptide comprises a sequence selectedfrom the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 18, 20and 22; and (e) a nucleotide sequence comprising a sequence selectedfrom the group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16,17, 19, 21, 23-26; and wherein said plant exhibits increased droughttolerance or improved nitrogen utilization when compared to a controlplant not comprising said polynucleotide.
 6. The plant of claim 5,wherein said plant exhibits said increase in yield when compared, underwater limiting conditions, to said control plant not comprising saidrecombinant DNA construct.
 7. The plant of any one of claim 5 whereinthe plant is a monocot.
 8. The plant of claim 5 wherein the plant isselected from the group consisting of: maize, soybean, sunflower,sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet,sugarcane, switchgrass, tobacco, potato and sugar beet.
 9. An isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding a polypeptide withdrought tolerance activity, wherein the polypeptide has an amino acidsequence of at least 90% sequence identity, based on the Clustal Wmethod of alignment with pairwise alignment default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=0.2, DELAY DEVERGENT SEQS(%)=30%, DNATRANSITION WEIGHT=0.5, PROTEIN WEIGHT MATRIX “Gonnet Series”), whencompared to a sequence selected from the group consisting of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; (b) a nucleotide sequenceencoding a polypeptide with drought tolerance activity, wherein thenucleotide sequence is hybridizable under stringent conditions with aDNA molecule comprising the full complement of SEQ ID NO a sequenceselected from the group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12,14, 16, 17, 19, 21, 23-26; (c) a nucleotide sequence encoding apolypeptide with drought tolerance activity, wherein the nucleotidesequence is a sequence selected from the group consisting of SEQ ID NOS:1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26 a sequence selectedfrom the group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16,17, 19, 21, 23-26; by alteration of one or more nucleotides by at leastone method selected from the group consisting of: deletion,substitution, addition and insertion; (d) a nucleotide sequence encodinga polypeptide wherein the amino acid sequence of the polypeptidecomprises a sequence selected from the group consisting of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 18, 20 and 22; and (e) a nucleotide sequencecomprising a sequence selected from the group consisting of SEQ ID NOS:1, 2, 4, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23-26 a sequence selectedfrom the group consisting of SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, 16,17, 19, 21, 23-26; wherein the polynucleotide is operably linked to aheterologous regulatory element.
 10. The polynucleotide of claim 9,wherein the polypeptide of part (a) has an amino acid sequence of atleast 80% sequence identity, based on the Clustal W method of alignmentwith the pairwise alignment default parameters, when compared to asequence selected from the group consisting of SEQ ID NOS: 3, 5, 7, 9,11, 13, 15, 18, 20 and
 22. 11. The polynucleotide of claim 9, whereinthe polypeptide of part (a) has an amino acid sequence of at least 85%sequence identity, based on the Clustal W method of alignment with thepairwise alignment default parameters, when compared to a sequenceselected from the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13,15, 18, 20 and
 22. 12. The polynucleotide of claim 9, wherein thepolypeptide of part (a) has an amino acid sequence of at least 90%sequence identity, based on the Clustal W method of alignment with thepairwise alignment default parameters, when compared to a sequenceselected from the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13,15, 18, 20 and
 22. 13. The polynucleotide of claim 9, wherein thepolypeptide of part (a) has an amino acid sequence of at least 95%sequence identity, based on the Clustal W method of alignment with thepairwise alignment default parameters, when compared to a sequenceselected from the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13,15, 18, 20 and
 22. 14. A recombinant DNA construct comprising theisolated polynucleotide of claim 9 operably linked to at least oneregulatory element.
 15. A cell comprising the recombinant DNA constructof claim 14, wherein the cell is selected from the group consisting of abacterial cell, a yeast cell, and insect cell and a plant cell.
 16. Aplant comprising the recombinant DNA construct of claim
 14. 17. A seedcomprising the recombinant DNA construct of claim
 14. 18. Seed of theplant of claim
 5. 19. The plant of claim 5, wherein the plant is maizeand wherein the polypeptide comprises an amino acid sequence of SEQ IDNO: 3 or a sequence that is at least 95% identical to SEQ ID NO: 3,wherein the plant shows one or more improved agronomic parameters thatcontribute to drought tolerance or yield.
 20. A maize plant cell derivedfrom the plant of claim 19.